B7-H4 antibodies and methods of use thereof

ABSTRACT

The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to human B7-H4 (and optionally cynomolgus monkey, mouse, and/or rat B7-H4) and compositions comprising such antibodies or antigen-binding fragments thereof. In a specific aspect, the antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4 increase T cell proliferation, increase interferon-gamma production, and/or deplete B7-H4 expressing cells via ADCC activity. The present disclosure also provides methods for treating disorders, such as cancer, by administering an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/550,173, filed Aug. 25, 2017, U.S. Provisional Application No.62/579,774, filed Oct. 31, 2017, U.S. Provisional Application No.62/607,810, filed Dec. 19, 2017, and U.S. Provisional Application No.62/656,789, filed Apr. 12, 2018, which are each hereby incorporated inits entirety by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:3986.0090004_SL_ST25.txt; Size: 331,354 bytes; Date of Creation: Aug. 3,2018) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION Field

The present disclosure relates to antibodies that specifically bind tohuman B7-H4 compositions comprising such antibodies, and methods ofproducing and using antibodies that specifically bind to B7-H4.

Description of the Related Art

B7-H4 (also known as B7x, B7-S1, and VTCN1) is an immune regulatorymolecule that shares homology with other B7 family members, includePD-L1. It is a type I transmembrane protein comprised of both IgV andIgC ectodomains. While B7-H4 expression in healthy tissues is relativelylimited at the protein level, B7-H4 is expressed in several solid tumorssuch as gynecological carcinomas of the breast, ovary, and endometrium.Expression of B7-H4 in tumors tends to correlate with poor prognosis.The receptor for B7-H4 is unknown, but it is believed to be expressed onT cells. B7-H4 is believed to directly inhibit T cell activity.

Given the expression and function of B7-H4, provided herein areantibodies that specifically bind to B7-H4 and the use of theseantibodies to modulate B7-H4 activity, including, e.g., in the treatmentof cancer.

SUMMARY

Provided herein is an isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4, comprising heavy chainvariable region (VH) complementarity determining region (CDR) 1, VHCDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3sequences selected from the group consisting of: SEQ ID NOs:5-10,respectively; SEQ ID NOs:15-20, respectively; SEQ ID NOs:25-30,respectively; SEQ ID NOs:35-40, respectively; SEQ ID NOs:458-463,respectively; SEQ ID NOs:45-50, respectively; SEQ ID NOs:55-60,respectively; SEQ ID NOs:65-70, respectively; SEQ ID NOs:75-80,respectively; SEQ ID NOs:85-90, respectively; SEQ ID NOs:95-100,respectively; SEQ ID NOs:105-110, respectively; SEQ ID NOs:115-120,respectively; SEQ ID NOs:125-130, respectively; SEQ ID NOs:135-140,respectively; SEQ ID NOs:145-150, respectively; SEQ ID NOs:155-160,respectively; SEQ ID NOs:165-170, respectively; SEQ ID NOs:175-180,respectively; SEQ ID NOs:185-190, respectively; and SEQ ID NOs:195-200,respectively.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a VH comprising the amino acid sequence of SEQ ID NO:11, 21,31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171,181, 191, or 201.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a VL comprising the amino acid sequence of SEQ ID NO:12, 22,32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182,192, or 202.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises the amino acidsequence of SEQ ID NO:11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111,121, 131, 141, 151, 161, 171, 181, 191, or 201.

Also provided herein is an isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4, wherein the antibodycomprises a heavy chain variable region and a light chain variableregion, wherein the light chain variable region comprises the amino acidsequence of SEQ ID NO:12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122,132, 142, 152, 162, 172, 182, 192, or 202.

Also provided herein is an isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4, comprising a heavy chainvariable region and a light chain variable region comprising the aminoacid sequences of: SEQ ID NOs:11 and 12, respectively; SEQ ID NOs:21 and22, respectively; SEQ ID NOs:31 and 32, respectively; SEQ ID NOs:41 and42, respectively; SEQ ID NOs:464 and 42, respectively, SEQ ID NOs:51 and52, respectively; SEQ ID NOs:61 and 62, respectively; SEQ ID NOs:71 and72, respectively; SEQ ID NOs:81 and 82, respectively; SEQ ID NOs:91 and92, respectively; SEQ ID NOs:101 and 102, respectively; SEQ ID NOs:111and 112, respectively; SEQ ID NOs:121 and 122, respectively; SEQ IDNOs:131 and 132, respectively; SEQ ID NOs:141 and 142, respectively; SEQID NOs:151 and 152, respectively; SEQ ID NOs:161 and 162, respectively;SEQ ID NOs:171 and 172, respectively; SEQ ID NOs:181 and 182,respectively; SEQ ID NOs:191 and 192, respectively; or SEQ ID NOs:201and 202, respectively.

In one embodiment, the antibody or antigen-binding fragment thereoffurther comprises a heavy chain constant region. In one embodiment, theheavy chain constant region is selected from the group consisting ofhuman immunoglobulins IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂ heavy chainconstant regions.

In one embodiment, the antibody or antigen-binding fragment furthercomprises a light chain constant region. In one embodiment, the lightchain constant region is selected from the group consisting of humanimmunoglobulins IgGκ and IgGλ light chain constant regions.

In one embodiment, the antibody or antigen-binding fragment furthercomprises a heavy chain constant region and a light chain constantregion, wherein the heavy chain constant region is a human IgG₁ heavychain constant region, and wherein the light chain constant region is ahuman IgGκ light chain constant region.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:13, 23, 33, 43, 469, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143,153, 163, 173, 183, 193, or 203.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a light chain comprising the amino acid sequence of SEQ IDNO:14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154,164, 174, 184, 194, or 204.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a heavy chain and light chain comprising the amino acidsequences of: SEQ ID NOs:13 and 14, respectively; SEQ ID NOs:23 and 24,respectively; SEQ ID NOs:33 and 34, respectively; SEQ ID NOs:43 and 44,respectively; SEQ ID NOs:469 and 44, respectively; SEQ ID NOs:53 and 54,respectively; SEQ ID NOs:63 and 64, respectively; SEQ ID NOs:73 and 74,respectively; SEQ ID NOs:83 and 84, respectively; SEQ ID NOs:93 and 94,respectively; SEQ ID NOs:103 and 104, respectively; SEQ ID NOs:113 and114, respectively; SEQ ID NOs:123 and 124, respectively; SEQ ID NOs:133and 134, respectively; SEQ ID NOs:143 and 144, respectively; SEQ IDNOs:153 and 154, respectively; SEQ ID NOs:163 and 164, respectively; SEQID NOs:173 and 174, respectively; SEQ ID NOs:183 and 184, respectively;SEQ ID NOs:193 and 194, respectively; or SEQ ID NOs:203 and 204,respectively.

Also provided herein is an isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4, wherein the antibody orantigen-binding fragment thereof comprises the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 of an antibody selected from thegroup consisting of 15461, 20500, 20501, 20502, 20502.1, 22208, 15462,22213, 15465, 20506, 15483, 20513, 22216, 15489, 20516, 15472, 15503,15495, 15478, 15441, and 20496. In one embodiment, the CDRs are theKabat-defined CDRs, the Chothia-defined CDRs, or the AbM-defined CDRs.

Also provided herein is an isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4, comprising the heavychain variable region (VH) complementarity determining region (CDR) 1,VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, andCDR3 sequences of SEQ ID NOs:458-463, respectively. In one embodiment,(i) the antibody or antigen-binding fragment thereof comprises avariable heavy chain region comprising the amino acid sequence of SEQ IDNO:464 and a variable light chain region comprising the amino acidsequence of SEQ ID NO:42 or (ii) the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:469 and a light chaincomprising the amino acid sequence of SEQ ID NO:44.

Also provided herein is an isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4, comprising the heavychain variable region (VH) complementarity determining region (CDR) 1,VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, andCDR3 sequences of SEQ ID NOs:35-40, respectively. In one embodiment, (i)the antibody or antigen-binding fragment thereof comprises a variableheavy chain region comprising the amino acid sequence of SEQ ID NO:41and a variable light chain region comprising the amino acid sequence ofSEQ ID NO:42 or (ii) the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:43 and a light chain comprising theamino acid sequence of SEQ ID NO:44.

Also provided herein is an isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4, comprising the heavychain variable region (VH) complementarity determining region (CDR) 1,VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, andCDR3 sequences of SEQ ID NOs:65-70, respectively. In one embodiment, (i)the antibody or antigen-binding fragment thereof comprises a variableheavy chain region comprising the amino acid sequence of SEQ ID NO:71and a variable light chain region comprising the amino acid sequence ofSEQ ID NO:72 or (ii) wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:73 and a light chaincomprising the amino acid sequence of SEQ ID NO:74.

Also provided herein is an isolated antibody or antigen-binding fragmentthereof that binds to the same epitope of human B7-H4 as the antibody orantigen-binding fragment thereof of any one of claims 1-20. In oneembodiment, the antibody or antigen-binding fragment thereof binds tothe same epitope of human B7-H4 as determined by SPR.

In one embodiment, the antibody or antigen-binding fragment thereof is ahuman antibody or antigen-binding fragment thereof. In one embodiment,the antibody or antigen-binding fragment thereof is a murine, humanized,or chimeric antibody or antigen-binding fragment thereof.

In one embodiment, the antibody or antigen-binding fragment thereofinduces T cell proliferation. In one embodiment, the antibody orantigen-binding fragment increases T-cell proliferation by at least 21%as compared to treatment with a control antibody. In one embodiment, theantibody or antigen-binding fragment increases T-cell proliferation byabout 5% to about 35% as compared to treatment with a control antibody.In one embodiment, the antibody or antigen-binding fragment thereofinduces CD4+ T cell proliferation. In one embodiment, the antibody orantigen-binding fragment thereof increases CD4+ T cell proliferation byat least 9% as compared to treatment with a control antibody. In oneembodiment, the antibody or antigen-binding fragment thereof increasesCD4+ T cell proliferation by about 5% to about 15% as compared totreatment with a control antibody. In one embodiment, the antibody orantigen-binding fragment thereof induces CD8+ T cell proliferation. Inone embodiment, the antibody or antigen-binding fragment thereofincreases CD8+ T cell proliferation by at least 11% as compared totreatment with a control antibody. In one embodiment, the antibody orantigen-binding fragment thereof increases CD8+ T cell proliferation byabout 5% to about 15% as compared to treatment with a control antibody.

In one embodiment, the antibody or antigen-binding fragment thereofinduces interferon-gamma (IFNγ) production. In one embodiment, theantibody or antigen-binding fragment thereof is capable of increasingproduction of IFNγ by at least 2 fold, at least 3 fold, at least 4 fold,and at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold,about 2 fold to about 10 fold, or about 3 fold to about 10 fold.

In one embodiment, the antibody or antigen-binding fragment thereof iscapable of inducing antibody dependent cell mediated cytotoxicity (ADCC)in a B7-H4-expressing cell. In one embodiment, the antibody orantigen-binding fragment thereof induces specific lysis in at least 20%,at least 30%, at least 40%, about 20% to about 50%, or about 30% toabout 50% of B7-H4 expressing cells.

In one embodiment, the antibody or antigen-binding fragment thereofinhibits tumor growth in a murine CT26 colorectal carcinoma model, amurine breast carcinoma 4T1 model, or a melanoma cell lineB16-moB7-H4/H3 model. In one embodiment, the antibody or antigen-bindingfragment thereof reduces tumor growth by at least 25%, at least 30%, atleast 40%, at least 45%, or at least 50% as compared to treatment with acontrol antibody.

In one embodiment, the induction of T cell proliferation, the inductionof CD4+ T cell proliferation, the induction of CD8+ T cellproliferation, the induction of IFNγ production, the ADCC activity,and/or in the inhibition of tumor growth is dose-dependent.

In one embodiment, the antibody or antigen-binding fragment thereofbinds to cynomolgus monkey B7-H4. In one embodiment, the antibody orantigen-binding fragment thereof binds to rat B7-H4. In one embodiment,the antibody or antigen-binding fragment thereof binds to mouse B7-H4.In one embodiment, the antibody or antigen-binding fragment thereofbinds to human B7-H4, cynomolgus monkey B7-H4, rat B7-H4 and mouseB7-H4.

In one embodiment, the antibody or antigen-binding fragment thereofbinds to the IgV domain of human B7-H4.

In one embodiment, the antibody or antigen-binding fragment thereof isafucosylated.

In one embodiment, the antibody or antigen binding fragment thereof is afull length antibody. In one embodiment, the antibody or antigen bindingfragment thereof is an antigen binding fragment. In one embodiment, theantigen binding fragment comprises a Fab, Fab′, F(ab)₂, single chain Fv(scFv), disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2,minibody, F(ab′)₃, tetrabody, triabody, diabody, single-domain antibody,DVD-Ig, Fcab, mAb², (scFv)₂, or scFv-Fc.

In one embodiment, the antibody or antigen-binding fragment thereoffurther comprises a detectable label.

Also provided herein is an isolated polynucleotide comprising a nucleicacid molecule encoding the heavy chain variable region or heavy chain ofan antibody or antigen-biding fragment thereof provided herein. In oneembodiment, the nucleic acid molecule encodes the VH of SEQ ID NO:11,21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161,171, 181, 191, or 201 or the heavy chain of SEQ ID NO:13, 23, 33, 43,469, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183,193, or 203. In one embodiment, the nucleic acid molecule comprises thesequence of SEQ ID NO:213, 223, 233, 243, 470, 253, 263, 273, 283, 293,303, 313, 323, 333, 343, 353, 363, 373, 383, 393, or 403. In oneembodiment, the nucleic acid molecule comprises (i) the sequence of SEQID NO:213, 223, 233, 243, 470, 253, 263, 273, 283, 293, 303, 313, 323,333, 343, 353, 363, 373, 383, 393, or 403 and (ii) the sequence of SEQID NO:408.

Also provided herein is an isolated polynucleotide comprising a nucleicacid molecule encoding the light chain variable region or light chain ofan antibody or antigen-biding fragment thereof provided herein. In oneembodiment, the nucleic acid molecule encodes the VL of SEQ ID NO:12,22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172,182, 192, or 202 or the light chain of SEQ ID NO:14, 24, 34, 44, 54, 64,74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, or 204. Inone embodiment, the nucleic acid molecule comprises the sequence of SEQID NO:214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334,344, 354, 364, 374, 384, 394, or 404. In one embodiment, the nucleicacid molecule comprises (i) the sequence of SEQ ID NO:214, 224, 234,244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374,384, 394, or 404 and (ii) the sequence of SEQ ID NO:406.

Also provided herein is an isolated polynucleotide comprising a nucleicacid molecule encoding the heavy chain variable region or heavy chain ofan antibody or antigen-biding fragment provided herein and the lightchain variable region or light chain of the antibody or antigen-bidingfragment thereof.

Also provided herein is an isolated vector comprising a polynucleotideprovided herein.

Also provided herein is a host cell comprising a polynucleotide providedherein, a vector provided herein, a first vector comprising onepolynucleotide provided herein (e.g., a polynucleotide comprising avariable heavy chain or heavy chain encoding nucleic acid) and a secondvector comprising another polynucleotide provided herein (e.g., apolynucleotide comprising variable light chain or light chain encodingnucleic acid). In one embodiment, the host cell is a cell selected fromthe group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces,yeast, CHO, YB/20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2,SP2/0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cell, plantcell, insect cell, and human cell in tissue culture. In one embodiment,the host cell is a CHO cell. In one embodiment, the host cell (e.g., amammalian host cell, such as a CHO cell) lacks a functionalalpha-1,6-fucosyltransferase gene (FUT8) gene.

Also provided herein is a method (e.g., an in vitro method) of producingan antibody or antigen-binding fragment thereof that binds to humanB7-H4 comprising culturing a host cell provided herein so that thenucleic acid molecule is expressed and the antibody or antigen-bidingfragment thereof is produced.

Also provided herein is an isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4 and is encoded by apolynucleotide provided herein.

Also provided herein is a pharmaceutical composition comprising anantibody or antigen-binding fragment thereof provided herein, apolynucleotide provided herein, a vector provided herein, or a host cellprovided herein; and a pharmaceutically acceptable excipient.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments provided herein and (ii) apharmaceutically acceptable excipient, wherein at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% of the antibodies or antigen-binding fragmentsthereof in the composition are afucosylated.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments provided herein and (ii) apharmaceutically acceptable excipient, wherein at least 95% of theantibodies or antigen-binding fragments thereof in the composition areafucosylated.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments thereof that specifically bindto human B7-H4 and comprise the heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ IDNOs:458-463, respectively and (ii) a pharmaceutically acceptableexcipient, wherein at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% of theantibodies or antigen-binding fragments thereof in the composition areafucosylated. In one embodiment, (i) the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region comprising theamino acid sequence of SEQ ID NO:464 and a variable light chain regioncomprising the amino acid sequence of SEQ ID NO:42 or (ii) the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:469 and a light chain comprising the amino acid sequence of SEQ IDNO:44.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments thereof that specifically bindto human B7-H4 and comprise the heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:458-463, respectively and (ii) a pharmaceutically acceptable excipient,wherein at least 95% of the antibodies or antigen-binding fragmentsthereof in the composition are afucosylated. In one embodiment, (i) theantibody or antigen-binding fragment thereof comprises a variable heavychain region comprising the amino acid sequence of SEQ ID NO: 464 and avariable light chain region comprising the amino acid sequence of SEQ IDNO:42 or (ii) the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 469 and a light chain comprising the aminoacid sequence of SEQ ID NO:44.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments thereof that specifically bindto human B7-H4 and comprise the heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ IDNOs:35-40, respectively and (ii) a pharmaceutically acceptableexcipient, wherein at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% of theantibodies or antigen-binding fragments thereof in the composition areafucosylated. In one embodiment, (i) the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region comprising theamino acid sequence of SEQ ID NO:41 and a variable light chain regioncomprising the amino acid sequence of SEQ ID NO:42 or (ii) the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:43 and a light chain comprising the amino acid sequence of SEQ IDNO:44.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments thereof that specifically bindto human B7-H4 and comprise the heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ IDNOs:35-40, respectively and (ii) a pharmaceutically acceptableexcipient, wherein at least 95% of the antibodies or antigen-bindingfragments thereof in the composition are afucosylated. In oneembodiment, (i) the antibody or antigen-binding fragment thereofcomprises a variable heavy chain region comprising the amino acidsequence of SEQ ID NO:41 and a variable light chain region comprisingthe amino acid sequence of SEQ ID NO:42 or (ii) the antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:43 and alight chain comprising the amino acid sequence of SEQ ID NO:44.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments thereof that specifically bindto human B7-H4 and comprise the heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ IDNOs:65-70, respectively and (ii) a pharmaceutically acceptableexcipient, wherein at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% of theantibodies or antigen-binding fragments thereof in the composition areafucosylated. In one embodiment, (i) the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region comprising theamino acid sequence of SEQ ID NO:71 and a variable light chain regioncomprising the amino acid sequence of SEQ ID NO:72 or (ii) wherein theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:73 and a light chain comprising the amino acid sequence of SEQID NO:74.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments thereof that specifically bindto human B7-H4 and comprise the heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ IDNOs:65-70, respectively and (ii) a pharmaceutically acceptableexcipient, wherein at least 95% of the antibodies or antigen-bindingfragments thereof in the composition are afucosylated. In oneembodiment, (i) the antibody or antigen-binding fragment thereofcomprises a variable heavy chain region comprising the amino acidsequence of SEQ ID NO:71 and a variable light chain region comprisingthe amino acid sequence of SEQ ID NO:72 or (ii) wherein the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:73 and a light chain comprising the amino acid sequence of SEQ IDNO:74.

In one embodiment, fucosylation is undetectable in the composition.

Also provided herein is a method for inducing T cell proliferationcomprising contacting a T cell with an antibody or antigen-bindingfragment thereof provided herein, a polynucleotide provided herein, avector provided herein, a host cell provided herein, or a pharmaceuticalcomposition provided herein. In one embodiment, T cell proliferation isreduced by at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, or at least 80% (e.g., as compared to treatment with acontrol antibody).

Also provided herein is a method for inducing CD4+ T cell proliferationcomprising contacting a CD4+ T cell with an antibody or antigen-bindingfragment thereof provided herein, a polynucleotide provided herein, avector provided herein, a host cell provided herein, or a pharmaceuticalcomposition provided herein. In one embodiment, CD4+ T cellproliferation is reduced by at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, or at least 80% (e.g., as compared to treatmentwith a control antibody).

Also provided herein is a method for inducing CD8+ T cell proliferationcomprising contacting a CD8+ T cell with an antibody or antigen-bindingfragment thereof provided herein, a polynucleotide provided herein, avector provided herein, a host cell provided herein, or a pharmaceuticalcomposition provided herein. In one embodiment, CD8+ T cellproliferation is reduced by at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, or at least 80% (e.g., as compared to treatmentwith a control antibody).

Also provided herein is a method for inducing interferon gammaproduction comprising contacting a T cell with an antibody orantigen-binding fragment thereof provided herein, a polynucleotideprovided herein, a vector provided herein, a host cell provided herein,or a pharmaceutical composition provided herein. In one embodiment,interferon gamma production is increased by at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, or at least 80% (e.g., ascompared to treatment with a control antibody).

Also provided herein is a method for killing a cell expressing B7-H4comprising contacting the cell with an antibody or antigen-bindingfragment thereof provided herein, a polynucleotide provided herein, avector provided herein, a host cell provided herein, or a pharmaceuticalcomposition provided herein.

Also provided herein is a method for depleting B7-H4-expressing cellsfrom a population of cells comprising contacting the population of cellswith an antibody or antigen-binding fragment thereof provided herein, apolynucleotide provided herein, a vector provided herein, a host cellprovided herein, or a pharmaceutical composition provided herein.

In one embodiment, the killing or depletion occurs via ADCC.

In one embodiment, the contacting is in vitro. In one embodiment, thecontacting is in a subject.

Also provided herein is a method of treating cancer in a subject, themethod comprising administering to the subject an effective amount of anantibody or antigen-binding fragment thereof provided herein, apolynucleotide provided herein, a vector provided herein, a host cellprovided herein, or a pharmaceutical composition provided herein. In oneembodiment, the cancer is selected from the group consisting of breastcancer, such as triple negative breast cancer or invasive ductalcarcinoma, endometrial carcinoma, ovarian cancer, non-small cell lungcancer, pancreatic cancer, thyroid cancer, kidney cancer and bladdercancer. In one embodiment, the breast cancer is triple negative breastcancer or wherein the non-small cell lung cancer is squamous cellcarcinoma. In one embodiment, the non-small cell lung cancer is anadenocarcinoma. In one embodiment, the cancer is selected from the groupconsisting of head and neck cancer, small cell lung cancer, gastriccancer, and melanoma. In one embodiment, the ovarian cancer is a serousadenocarcinoma. In one embodiment, the breast cancer is a ductaladenocarcinoma.

In one embodiment, the cancer is a PD-1 inhibitor inadequate responderand/or PD-L1 inhibitor inadequate responder. In one embodiment, thecancer expresses a low level of PD-L1.

In one embodiment, the subject is human.

Also provided herein is a method for detecting B7-H4 in a samplecomprising contacting said sample with an antibody or antigen-bindingfragment thereof provided herein. In one embodiment, the sample isobtained from a cancer in a human subject.

Also provided herein is a kit comprising an antibody or antigen-bindingfragment thereof provided herein, a polynucleotide provided herein, avector provided herein, a host cell provided herein, or a pharmaceuticalcomposition provided herein and a) a detection reagent, b) a B7-H4antigen, c) a notice that reflects approval for use or sale for humanadministration, or d) a combination thereof.

Also provided herein is an isolated antibody or antigen-bindingfragment, wherein the antibody or antigen-binding fragment thereofinhibits T cell checkpoint blockade activity of B7-H4. In oneembodiment, the T cell checkpoint blockade activity is measured by anincrease in IL-2 production relative to control cells.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments and (ii) a pharmaceuticallyacceptable excipient, wherein the antibodies or antigen-bindingfragments inhibit T cell checkpoint blockade activity of B7-H4. In anembodiment, the T cell checkpoint blockade activity is measured by anincrease in IL-2 production relative to control cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows B7-H4 expression in a variety of tumor tissues. (SeeExample 1.)

FIG. 1B shows B7-H4 prevalence in various tumor types by IHC. (SeeExample 1.)

FIG. 2A shows the binding of B7-H4 antibodies to HEK293T cellsexpressing human, cynomolgus monkey, or mouse B7-H4. (See Example 6.)

FIG. 2B shows the binding of B7-H4 antibodies to SK-BR-3 cells and toHEK293T cells expressing mouse, cynomolgus monkey, or rat B7-H4. (SeeExample 6.)

FIG. 3A shows the effect of B7-H4 antibodies on CD4⁺, CD8⁺ or total Tcell proliferation. (See Example 7.)

FIG. 3B shows the effect of B7-H4 antibodies on interferon-gamma (IFNγ)production. (See Example 7.)

FIG. 3C shows the effect of B7-H4 antibodies on CD4⁺, CD8⁺ or total Tcell proliferation and on interferon-gamma (IFNγ) production. (SeeExample 7.)

FIG. 3D shows the effect of various concentrations of B7-H4 antibodieson interferon-gamma (IFNγ) production. (See Example 7.)

FIGS. 4A-4D show the binding of fucosylated and afucosylated antibodiesto B7-H4 on SK-BR-3 cells (FIG. 4A) and to HEK293T cells expressingmouse (FIG. 4B), cynomolgus monkey (FIG. 4C), or rat B7-H4 (FIG. 4D).(See Example 9.)

FIGS. 5A and 5B show the binding of afucosylated and fucosylated B7-H4antibodies (respectively) to human Fcγ receptor IIIa (FcγRIIIa) V158allele. (See Example 10.)

FIG. 6A shows the T cell checkpoint blockade activity of fucosylated andafucosylated B7-H4 antibodies. (See Example 11.)

FIG. 6B shows T cell checkpoint ligand activity of afucosylated B7-H4antibodies compared to isotype control treated cells as measured by IL-2production. (See Example 11.)

FIG. 7 shows the ADCC activity of fucosylated and afucosylated B7-H4antibodies against a B7-H4 expressing cell line. (See Example 12.)

FIG. 8 shows the ADCC activity of fucosylated and afucosylated B7-H4antibodies against cells with various B7-H4 expression levels. (SeeExample 13.)

FIG. 9A shows the in vivo anti-tumor efficacy of the B7-H4 antibody20502. (See Example 14.)

FIG. 9B shows the in vivo anti-tumor efficacy of the B7-H4 antibody22213. (See Example 14.)

FIG. 10 shows the in vivo anti-tumor efficacy of a murine version of theB7-H4 antibody 20502 in mice implanted with 4T1 (breast carcinoma) andB16 (melanoma) cells. (See Example 14.)

FIG. 11 shows the in vivo anti-tumor efficacy of afucosylated 20502 inan MX-1 human breast cancer xenograft model. The images show human B7-H4expression in the MX-1 xenograft tumor (left) and murine B7-H4expression in the 4T1 tumors (right). (See Example 14.)

FIGS. 12A-12C show the in vivo anti-tumor efficacy of 20502-msIgG2a-Fadministered on the same day as an anti-PD-1 antibody. (See Example15.) * indicates p<0.05; and **** indicates p<0.0001.

FIG. 13 shows that treatment with afucosylated 20502 (bottom panels)results in NK cell infiltration (left panels), T-cell infiltration(center panels), and PD-L1 up-regulation (right panels) as compared totreatment with a control antibody (top panels). (See Example 16.)

FIG. 14A shows that 20502-msIgG2a-F antibody significantly reduces tumorgrowth in 4T1 breast carcinoma cells in a dose dependent manner. (SeeExample 17.)

FIG. 14B shows that 20502-msIgG2a-F antibody significantly inhibitstumor growth at 30 mg/kg (p=0.0003), 20 mg/kg (p=0.0103), 10 mg/kg(p=0.0419), 3 mg/kg (p=0.0277), and 1 mg/kg (p=0.0333) as assessed byOneWay ANOVA on Day 30. (See Example 17.)

FIG. 15A shows that 20502-msIgG2a-F antibody significantly reducesgrowth of B16 expressing B7-H4/H3. (See Example 17.)

FIG. 15B shows that 20502-msIgG2a-F antibody significantly inhibitstumor growth at 30 mg/kg (p=0.0085), 20 mg/kg (p=0.0041), 10 mg/kg(p=0.0017), and 3 mg/kg (p=0.0420) as assessed by OneWay ANOVA on Day23. (See Example 17.)

DETAILED DESCRIPTION

Provided herein are antibodies (e.g., monoclonal antibodies) andantigen-binding fragments thereof that specifically bind to B7-H4 (e.g.,human B7-H4). The anti-B7-H4 antibodies and antigen-binding fragmentsthereof can, for example, result in T cell checkpoint blockade activity(e.g., as measured by an increase in interferon-gamma (IFNγ), CD4 T-cellproliferation, CD8 T-cell proliferation, and/or total T-cellproliferation) and/or have antibody-dependent cellular cytotoxicity(ADCC activity).

Also provided are isolated nucleic acids (polynucleotides), such ascomplementary DNA (cDNA), encoding such antibodies and antigen-bindingfragments thereof. Further provided are vectors (e.g., expressionvectors) and cells (e.g., host cells) comprising nucleic acids(polynucleotides) encoding such antibodies and antigen-binding fragmentsthereof. Also provided are methods of making such antibodies andantigen-binding fragments thereof. In other aspects, provided herein aremethods for treating certain conditions, such as cancer. Relatedcompositions (e.g., pharmaceutical compositions), kits, and detectionmethods are also provided.

1.1 Terminology

As used herein, the term “B7-H4” refers to mammalian B7-H4 polypeptidesincluding, but not limited to, native B7-H4 polypeptides and isoforms ofB7-H4 polypeptides. “B7-H4” encompasses full-length, unprocessed B7-H4polypeptides as well as forms of B7-H4 polypeptides that result fromprocessing within the cell. As used herein, the term “human B7-H4”refers to a polypeptide comprising the amino acid sequence of SEQ IDNO:1. A “B7-H4 polynucleotide,” “B7-H4 nucleotide,” or “B7-H4 nucleicacid” refer to a polynucleotide encoding B7-H4.

The term “PD-1” as used herein, refers to mammalian PD-1 polypeptides,including, but not limited to, native PD-1 polypeptides and isoforms ofPD-1 polypeptides. PD-1 is also referred to as Programmed death protein1 or Programmed cell death protein 1. “PD-1” encompasses full-length,unprocessed PD-1 polypeptides as well as forms of PD-1 polypeptides thatresult from processing in the cell. As used herein, the term “humanPD-1” refers to a polypeptide comprising the amino acid sequence of SEQID NO:439: PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRMSPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAPKAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAVICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYATIVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO:439) (mature humanPD-1 without a signal sequence). A “PD-1 polynucleotide,” “PD-1nucleotide” or “PD-1 nucleic acid” refers to a polynucleotide encodingPD-1.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antibody, and any other modified immunoglobulinmolecule so long as the antibodies exhibit the desired biologicalactivity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

The term “antibody fragment” refers to a portion of an intact antibody.An “antigen-binding fragment,” “antigen-binding domain,” or“antigen-binding region,” refers to a portion of an intact antibody thatbinds to an antigen. An antigen-binding fragment can contain theantigenic determining regions of an intact antibody (e.g., thecomplementarity determining regions (CDR)). Examples of antigen-bindingfragments of antibodies include, but are not limited to Fab, Fab′,F(ab′)2, and Fv fragments, linear antibodies, and single chainantibodies. An antigen-binding fragment of an antibody can be derivedfrom any animal species, such as rodents (e.g., mouse, rat, or hamster)and humans or can be artificially produced.

The terms “anti-B7-H4 antibody,” “B7-H4 antibody” and “antibody thatbinds to B7-H4” refer to an antibody that is capable of binding B7-H4with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting B7-H4. The extent ofbinding of an anti-B7-H4 antibody to an unrelated, non-B7-H4 protein canbe less than about 10% of the binding of the antibody to B7-H4 asmeasured, e.g., by a radioimmunoassay (RIA).

The terms “anti-PD-1 antibody,” “PD-1 antibody” and “antibody that bindsto PD-1” refer to an antibody that is capable of binding PD-1 withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting PD-1. The extent of binding of ananti-PD-1 antibody to an unrelated, non-PD-1 protein can be less thanabout 10% of the binding of the antibody to PD-1 as measured, e.g., by aradioimmunoassay (RIA).

A “monoclonal” antibody or antigen-binding fragment thereof refers to ahomogeneous antibody or antigen-binding fragment population involved inthe highly specific recognition and binding of a single antigenicdeterminant, or epitope. This is in contrast to polyclonal antibodiesthat typically include different antibodies directed against differentantigenic determinants. The term “monoclonal” antibody orantigen-binding fragment thereof encompasses both intact and full-lengthmonoclonal antibodies as well as antibody fragments (such as Fab, Fab′,F(ab′)2, Fv), single chain (scFv) mutants, fusion proteins comprising anantibody portion, and any other modified immunoglobulin moleculecomprising an antigen recognition site. Furthermore, “monoclonal”antibody or antigen-binding fragment thereof refers to such antibodiesand antigen-binding fragments thereof made in any number of mannersincluding but not limited to by hybridoma, phage selection, recombinantexpression, and transgenic animals.

As used herein, the terms “variable region” or “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 120amino acids or 110 to 125 amino acids in the mature heavy chain andabout 90 to 115 amino acids in the mature light chain, which differextensively in sequence among antibodies and are used in the binding andspecificity of a particular antibody for its particular antigen. Thevariability in sequence is concentrated in those regions calledcomplementarity determining regions (CDRs) while the more highlyconserved regions in the variable domain are called framework regions(FR). Without wishing to be bound by any particular mechanism or theory,it is believed that the CDRs of the light and heavy chains are primarilyresponsible for the interaction and specificity of the antibody withantigen. In certain embodiments, the variable region is a human variableregion. In certain embodiments, the variable region comprises rodent ormurine CDRs and human framework regions (FRs). In particularembodiments, the variable region is a primate (e.g., non-human primate)variable region. In certain embodiments, the variable region comprisesrodent or murine CDRs and primate (e.g., non-human primate) frameworkregions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

The term “Kabat numbering” and like terms are recognized in the art andrefer to a system of numbering amino acid residues in the heavy andlight chain variable regions of an antibody or an antigen-bindingfragment thereof. In certain aspects, CDRs can be determined accordingto the Kabat numbering system (see, e.g., Kabat E A & Wu T T (1971) AnnNY Acad Sci 190: 382-391 and Kabat E A et al., (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242). Using the Kabatnumbering system, CDRs within an antibody heavy chain molecule aretypically present at amino acid positions 31 to 35, which optionally caninclude one or two additional amino acids, following 35 (referred to inthe Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using theKabat numbering system, CDRs within an antibody light chain molecule aretypically present at amino acid positions 24 to 34 (CDR1), amino acidpositions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). Ina specific embodiment, the CDRs of the antibodies described herein havebeen determined according to the Kabat numbering scheme.

Chothia refers instead to the location of the structural loops (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the ChothiaCDR-H1 loop when numbered using the Kabat numbering convention variesbetween H32 and H34 depending on the length of the loop (this is becausethe Kabat numbering scheme places the insertions at H35A and H35B; ifneither 35A nor 35B is present, the loop ends at 32; if only 35A ispresent, the loop ends at 33; if both 35A and 35B are present, the loopends at 34). The AbM hypervariable regions represent a compromisebetween the Kabat CDRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software.

Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32..34(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia Numbering) H2H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

As used herein, the term “constant region” or “constant domain” areinterchangeable and have its meaning common in the art. The constantregion is an antibody portion, e.g., a carboxyl terminal portion of alight and/or heavy chain which is not directly involved in binding of anantibody to antigen but which can exhibit various effector functions,such as interaction with the Fc receptor. The constant region of animmunoglobulin molecule generally has a more conserved amino acidsequence relative to an immunoglobulin variable domain. In certainaspects, an antibody or antigen-binding fragment comprises a constantregion or portion thereof that is sufficient for antibody-dependentcell-mediated cytotoxicity (ADCC).

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG, and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃, and IgG₄. Heavy chain amino acid sequences are wellknown in the art. In specific embodiments, the heavy chain is a humanheavy chain.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art. In specific embodiments,the light chain is a human light chain.

The term “chimeric” antibodies or antigen-binding fragments thereofrefers to antibodies or antigen-binding fragments thereof wherein theamino acid sequence is derived from two or more species. Typically, thevariable region of both light and heavy chains corresponds to thevariable region of antibodies or antigen-binding fragments thereofderived from one species of mammals (e.g. mouse, rat, rabbit, etc.) withthe desired specificity, affinity, and capability while the constantregions are homologous to the sequences in antibodies or antigen-bindingfragments thereof derived from another (usually human) to avoideliciting an immune response in that species.

The term “humanized” antibody or antigen-binding fragment thereof refersto forms of non-human (e.g. murine) antibodies or antigen-bindingfragments that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies orantigen-binding fragments thereof are human immunoglobulins in whichresidues from the complementary determining region (CDR) are replaced byresidues from the CDR of a non-human species (e.g. mouse, rat, rabbit,hamster) that have the desired specificity, affinity, and capability(“CDR grafted”) (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536(1988)). In some instances, the Fv framework region (FR) residues of ahuman immunoglobulin are replaced with the corresponding residues in anantibody or fragment from a non-human species that has the desiredspecificity, affinity, and capability. The humanized antibody orantigen-binding fragment thereof can be further modified by thesubstitution of additional residues either in the Fv framework regionand/or within the replaced non-human residues to refine and optimizeantibody or antigen-binding fragment thereof specificity, affinity,and/or capability. In general, the humanized antibody or antigen-bindingfragment thereof will comprise substantially all of at least one, andtypically two or three, variable domains containing all or substantiallyall of the CDR regions that correspond to the non-human immunoglobulinwhereas all or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody orantigen-binding fragment thereof can also comprise at least a portion ofan immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin. Examples of methods used to generate humanizedantibodies are described in U.S. Pat. No. 5,225,539; Roguska et al.,Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al.,Protein Eng. 9(10):895-904 (1996). In some embodiments, a “humanizedantibody” is a resurfaced antibody.

The term “human” antibody or antigen-binding fragment thereof means anantibody or antigen-binding fragment thereof having an amino acidsequence derived from a human immunoglobulin gene locus, where suchantibody or antigen-binding fragment is made using any technique knownin the art. This definition of a human antibody or antigen-bindingfragment thereof includes intact or full-length antibodies and fragmentsthereof.

An “afucosylated” antibody or antigen-binding fragment thereof or anantibody or antigen-binding fragment thereof “lacking fucose” refers toan IgG1 or IgG3 isotype antibody or antigen-binding fragment thereofthat lacks fucose in its constant region glycosylation. Glycosylation ofhuman IgG1 or IgG3 occurs at Asn297 as core fucosylated biantennarycomplex oligosaccharide glycosylation terminated with up to 2 Galresidues. In some embodiments, an afucosylated antibody lacks fucose atAsn297. These structures are designated as G0, G1 (a 1,6 or a 1,3), orG2 glycan residues, depending on the amount of terminal Gal residues.See, e.g., Raju, T. S., BioProcess Int. 1: 44-53 (2003). CHO typeglycosylation of antibody Fc is described, e.g., in Routier, F. FL,Glycoconjugate J. 14: 201-207 (1997).

Methods of measuring fucose include any methods known in the art. Forpurposes herein, fucose is detected by the method described in Example 1of WO2015/017600, which is herein incorporated by reference in itsentirety. Briefly, glycan analysis is performed by releasing glycansfrom the antibody (e.g., by enzymatic release), labeling the glycanswith anthranilic acid (2-AA), and then purifying the labeled glycans.Normal phase HPLC with fluorescent detection is used to separate theglycans and measure the relative amount of each glycan in the antibody.The glycans may be positively identified as lacking or including fucoseby mass spectrometry. In some embodiments, fucose is undetectable in acomposition comprising a plurality of afucosylated antibodies orantigen-binding fragments thereof. In some embodiments, an afucosylatedantibody or antigen-binding fragment thereof has enhanced ADCC activity,which may be measured by the assay provided in Example 12 herein. Insome embodiments, an afucosylated antibody or antigen-binding fragmentthereof has enhanced affinity for Fc gamma RIIIA. In some embodiments,an afucosylated antibody or antigen-binding fragment thereof hasenhanced affinity for Fc gamma RIIIA(V158). In some embodiments, anafucosylated antibody or antigen-binding fragment thereof has enhancedaffinity for Fc gamma RIIIA(F158). Affinity for Fc gamma RIIIA or itsalleles may be measure by the assay provided in Example 10 herein.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody or antigen-binding fragment thereof) and its bindingpartner (e.g., an antigen). Unless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a1:1 interaction between members of a binding pair (e.g., antibody orantigen-binding fragment thereof and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured and/or expressedin a number of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D)), and equilibrium associationconstant (K_(A)). The K_(D) is calculated from the quotient ofk_(off)/k_(on), whereas K_(A) is calculated from the quotient ofk_(on)/k_(off). k_(on) refers to the association rate constant of, e.g.,an antibody or antigen-binding fragment thereof to an antigen, andk_(off) refers to the dissociation of, e.g., an antibody orantigen-binding fragment thereof from an antigen. The k_(on) and k_(off)can be determined by techniques known to one of ordinary skill in theart, such as BIAcore® or KinExA.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody or antigen-bindingfragment thereof can specifically bind. An epitope can be, for example,contiguous amino acids of a polypeptide (linear or contiguous epitope)or an epitope can, for example, come together from two or morenon-contiguous regions of a polypeptide or polypeptides (conformational,non-linear, discontinuous, or non-contiguous epitope). In certainembodiments, the epitope to which an antibody or antigen-bindingfragment thereof binds can be determined by, e.g., NMR spectroscopy,X-ray diffraction crystallography studies, ELISA assays,hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquidchromatography electrospray mass spectrometry), array-basedoligo-peptide scanning assays, and/or mutagenesis mapping (e.g.,site-directed mutagenesis mapping). For X-ray crystallography,crystallization may be accomplished using any of the known methods inthe art (e.g., Giegé R et al., (1994) Acta Crystallogr D BiolCrystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) JBiol Chem 251: 6300-6303). Antibody/antigen-binding fragment thereof:antigen crystals can be studied using well known X-ray diffractiontechniques and can be refined using computer software such as X-PLOR(Yale University, 1992, distributed by Molecular Simulations, Inc.; see,e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H W et al.;U.S. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr DBiol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies can beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and CunninghamB C & Wells J A (1989) Science 244: 1081-1085 for a description ofmutagenesis techniques, including alanine scanning mutagenesistechniques.

A B7-H4 antibody that “binds to the same epitope” as a reference B7-H4antibody refers to an antibody that binds to the same B7-H4 amino acidresidues as the reference B7-H4 antibody. The ability of a B7-H4antibody to bind to the same epitope as a reference B7-4 antibody isdetermined by a hydrogen/deuterium exchange assay (see Coales et al.Rapid Commun. Mass Spectrom. 2009; 23: 639-647).

As used herein, the terms “immunospecifically binds,”“immunospecifically recognizes,” “specifically binds,” and “specificallyrecognizes” are analogous terms in the context of antibodies orantigen-binding fragments thereof. These terms indicate that theantibody or antigen-binding fragment thereof binds to an epitope via itsantigen-binding domain and that the binding entails some complementaritybetween the antigen binding domain and the epitope. Accordingly, anantibody that “specifically binds” to human B7-H4 (SEQ ID NO:1) may alsobind to B7-H4 from other species (e.g., cynomolgous monkey, mouse,and/or rat B7-H4) and/or B7-H4 proteins produced from other humanalleles, but the extent of binding to an un-related, non-B7-H4 protein(e.g., other B7 protein family members such as PD-L1) is less than about10% of the binding of the antibody to B7-H4 as measured, e.g., by aradioimmunoassay (RIA).

In a specific embodiment, provided herein is an antibody orantigen-binding fragment thereof that binds to human, cynomolgus monkey,mouse, and rat B7-H4.

An antibody is said to “competitively inhibit” binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeor an overlapping epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitionmay be determined by any method known in the art, for example,competition ELISA assays. An antibody may be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cell or compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some embodiments,an antibody, polynucleotide, vector, cell, or composition which isisolated is substantially pure. As used herein, “substantially pure”refers to material which is at least 50% pure (i.e., free fromcontaminants), at least 90% pure, at least 95% pure, at least 98% pure,or at least 99% pure.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

“Percent identity” refers to the extent of identity between twosequences (e.g., amino acid sequences or nucleic acid sequences).Percent identity can be determined by aligning two sequences,introducing gaps to maximize identity between the sequences. Alignmentscan be generated using programs known in the art. For purposes herein,alignment of nucleotide sequences can be performed with the blastnprogram set at default parameters, and alignment of amino acid sequencescan be performed with the blastp program set at default parameters (seeNational Center for Biotechnology Information (NCBI) on the worldwideweb, ncbi.nlm.nih.gov).

As used herein, the term “host cell” can be any type of cell, e.g., aprimary cell, a cell in culture, or a cell from a cell line. In specificembodiments, the term “host cell” refers to a cell transfected with anucleic acid molecule and the progeny or potential progeny of such acell. Progeny of such a cell may not be identical to the parent celltransfected with the nucleic acid molecule, e.g., due to mutations orenvironmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. The formulation can be sterile.

The terms “administer”, “administering”, “administration”, and the like,as used herein, refer to methods that may be used to enable delivery ofa drug, e.g., an anti-B7-H4 antibody or antigen-binding fragment thereofto the desired site of biological action (e.g., intravenousadministration). Administration techniques that can be employed with theagents and methods described herein are found in e.g., Goodman andGilman, The Pharmacological Basis of Therapeutics, current edition,Pergamon; and Remington's, Pharmaceutical Sciences, current edition,Mack Publishing Co., Easton, Pa.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) or consecutive administrationin any order.

The combination therapy can provide “synergy,” i.e., the effect achievedwhen the active agents used together is greater than the sum of theeffects that result from using the active agents separately. Asynergistic effect can be attained when the active agents are: (1)co-formulated and administered or delivered simultaneously in acombined, unit dosage formulation; (2) delivered serially, byalternation, or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the active agents are administered ordelivered sequentially, e.g., by different injections in separatesyringes. A “synergistic combination” produces an effect that is greaterthan the sum of the effects of the individual active agents of thecombination.

The combination therapy can provide an “additive” effect, i.e., theeffect achieved when the active agents used together is equal to the sumof the effects the result from using the active agents separately.

As used herein, the terms “subject” and “patient” are usedinterchangeably. The subject can be an animal. In some embodiments, thesubject is a mammal such as a non-human animal (e.g., cow, pig, horse,cat, dog, rat, mouse, monkey or other primate, etc.). In someembodiments, the subject is a cynomolgus monkey. In some embodiments,the subject is a human.

The term “therapeutically effective amount” refers to an amount of adrug, e.g., an anti-B7-H4 antibody or antigen-binding fragment thereofeffective to treat a disease or disorder in a subject. In the case ofcancer, the therapeutically effective amount of the drug can reduce thenumber of cancer cells; reduce the tumor size or burden; inhibit (i.e.,slow to some extent and in a certain embodiment, stop) cancer cellinfiltration into peripheral organs; inhibit (i.e., slow to some extentand in a certain embodiment, stop) tumor metastasis; inhibit, to someextent, tumor growth; relieve to some extent one or more of the symptomsassociated with the cancer; and/or result in a favorable response suchas increased progression-free survival (PFS), disease-free survival(DFS), or overall survival (OS), complete response (CR), partialresponse (PR), or, in some cases, stable disease (SD), a decrease inprogressive disease (PD), a reduced time to progression (TTP), or anycombination thereof. To the extent the drug can prevent growth and/orkill existing cancer cells, it can be cytostatic and/or cytotoxic.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to therapeutic measures that cure, slow down,lessen symptoms of, and/or halt progression of a diagnosed pathologiccondition or disorder. Thus, those in need of treatment include thosealready diagnosed with or suspected of having the disorder. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: a reduction in the number of or complete absence ofcancer cells; a reduction in the tumor size; inhibition of or an absenceof cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibition ofor an absence of tumor metastasis; inhibition or an absence of tumorgrowth; relief of one or more symptoms associated with the specificcancer; reduced morbidity and mortality; improvement in quality of life;reduction in tumorigenicity, tumorigenic frequency, or tumorigeniccapacity, of a tumor; reduction in the number or frequency of cancerstem cells in a tumor; differentiation of tumorigenic cells to anon-tumorigenic state; increased progression-free survival (PFS),disease-free survival (DFS), or overall survival (OS), complete response(CR), partial response (PR), stable disease (SD), a decrease inprogressive disease (PD), a reduced time to progression (TTP), or anycombination thereof.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. Examples of cancer include,but are not limited to, gynecological cancers (e.g., breast cancer(including triple negative breast cancer, ductal carcinoma), ovariancancer, and endometrial cancer), non-small cell lung cancer, pancreaticcancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), andbladder cancer (e.g., urothelial cell carcinoma). A non-small cell lungcancer can be e.g., an adenocarcinoma. Additional examples of cancerinclude, e.g., head and neck cancer, small cell lung cancer, gastriccancer, melanoma, cholangiocarcinoma, glioblastoma or glioblastomamultiforme (GBM), and merkel cell carcinoma. In one embodiment, theovarian cancer is a serous adenocarcinoma. In one embodiment, the breastcancer is a ductal adenocarcinoma. The cancer can be a “cancer thatexpresses B7-H4” or a “B7-H4 expressing cancer.” Such terms refer to acancer comprising cells that express B7-H4. The cancer may be a primarytumor or may be advanced or metastatic cancer.

A “refractory” cancer is one that progresses even though an anti-tumortreatment, such as a chemotherapy, is administered to the cancerpatient.

A “recurrent” cancer is one that has regrown, either at the initial siteor at a distant site, after a response to initial therapy.

A “relapsed” patient is one who has signs or symptoms of cancer afterremission. Optionally, the patient has relapsed after adjuvant orneoadjuvant therapy.

“T cell checkpoint blockade activity” refers to blocking or inhibitionof a T cell checkpoint activity or response. T cell checkpoint blockadeactivity can be measured in an artificial antigen presenting cells(aAPC) assay based on changes in IFNγ production. Primary human T cellscan be enriched from PBMCs using a T cell enrichment kit (such as anEasySep™ Human T Cell Enrichment Kit or similar kit). Enriched T cellsare incubated with beads (e.g., anti-CD3/anti-CD28 beads). After aperiod of time, the beads are magnetically removed, and T cells arewashed and incubated. Next, T cells are washed and incubated along withartificial antigen presenting cells (aAPCs) in the presence of B7-H4antibody dose titration. aAPCs can be treated with Mitomycin C and thenthoroughly washed prior to adding to the T cell co-culture. Afterco-culture of T cells, aAPCs, and B7-H4 antibodies, plates can becentrifuged, and supernatants can be harvested and assessed for IFNγproduction by ELISA. IFNγ production can be plotted vs. antibodyconcentration, and the EC50 potency can be calculated using nonlinearregression curve fit. Results can be measured as EC50+/− STD in nM. Tcell checkpoint blockade activity by B7-H4 antibodies can bedemonstrated by an increase in IFNγ production. “T cell checkpointblockade activity” can also be measured in an assay using cells thatendogenously express B7-H4. Primary human T cells can be enriched fromHLA-A2+ donor PBMCs using a T cell isolation kit (e.g., Human Pan T CellIsolation Kit). MART-I TCR expressing T cells can be generated by firstactivating enriched Pan T cells with beads (e.g., anti-CD3/anti-CD28Dynabeads), IL-2 and IL-7 for 48 hours. Activated T cells can then betransduced with MART-I TCR lentiviral particles in the presence of IL-2,IL-7 and polybrene. After transduction, MART-I TCR+ Pan T cells can beexpanded over a period of time in the presence of IL-2 and IL-7. Togenerate HLA-A2 expressing target cell lines, the endogenous B7-H4expressing cancer cell lines can be transduced with HLA-A2 lentiviralparticles for a period of time (e.g., 48 hours). Furthermore, B7-H4 canbe knocked-out of the HLA-A2+ cell line. Then, MART-I TCR+ Pan T cellscan be co-cultured in the presence of the various target cell lines at a1:1 E:T ratio, MART-I peptide and a B7-H4 antibody or a human isotypecontrol. After co-incubation, plates can be centrifuged, andsupernatants can be harvested and assessed for IL-2 production. IL-2production can be measured by a standard immunoassay kit (such as anAlphaLISA assay or similar assay).

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided. In this disclosure, “comprises,” “comprising,” “containing”and “having” and the like can have the meaning ascribed to them in U.S.Patent law and can mean “includes,” “including,” and the like;“consisting essentially of” or “consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. The term “and/or” as used in aphrase such as “A and/or B” herein is intended to include both “A andB,” “A or B,” “A,” and “B.” Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above and 5% to 10% below the value or range remain within theintended meaning of the recited value or range.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

1.2 Antibodies

In a specific aspect, provided herein are antibodies (e.g., monoclonalantibodies, such as chimeric, humanized, or human antibodies) andantigen-binding fragments thereof which specifically bind to B7-H4(e.g., human B7-H4). The amino acid sequences for human, cynomolgusmonkey, murine, and rat B7-H4 are known in the art and also providedherein as represented by SEQ ID NOs:1-4, respectively.

Human B7-H4: (SEQ ID NO: 1)MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMERGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK Cynomolgus monkey B7-H4: (SEQ ID NO: 2)MASLGQILFWSIISIIFILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVIGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFLAISWALLPLAPYLMLK Murine B7-H4 (SEQ ID NO: 3)MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIRTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLSCCLMLR Rat B7-H4 (SEQ ID NO: 4)MASLGQIIFWSIINVIIILAGAIVLIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIHTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLELLNSGPSPCVSSVSAAGWALLSLSCCLMLR

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4. In certain embodiments, anantibody or antigen-binding fragment thereof binds to human andcynomolgus monkey B7-H4. In certain embodiments, an antibody orantigen-binding fragment thereof binds to human, murine, and rat B7-H4.In certain embodiments, an antibody or antigen-binding fragment thereofbinds to human, cynomolgus monkey, murine, and rat B7-H4.

B7-H4 contains an IgC ectodomain (amino acids 153-241 of SEQ ID NO:1)and an IgV domain (amino acids 35-146 of SEQ ID NO:1).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to the IgV domain of human B7-H4. Accordingly,provided herein are antibodies and antigen-binding fragments thereofthat bind to a polypeptide consisting of amino acids 35-146 of SEQ IDNO:1.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2).

TABLE 1 VH CDR Amino Acid Sequences¹ VH CDR1 VH CDR2 VH CDR3 Antibody(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 15461 GSISSSSYYWGNIYYSGSTYYNPSLKS AREGSYPNWFDP (SEQ (SEQ ID NO: 5) (SEQ ID NO: 6)ID NO: 7) 20500 GSIKSGSHYWG NIYYSGSTYYNPSLRS AREGSYPNWFDP (SEQ(SEQ ID NO: 15) (SEQ ID NO: 16) ID NO: 17) 20501 GSIKSGSHYWGNIYYSGSTYYNPSLKS AREGSYPNWLDP (SEQ (SEQ ID NO: 25) (SEQ ID NO: 26)ID NO: 27) 20502 GSIKSGSYYWG NIYYSGSTYYNPSLRS AREGSYPNQFDP (SEQ(SEQ ID NO: 458) (SEQ ID NO: 459) ID NO: 460) 20502.1 GSIKSGSYYWGNIYYSGSTYYNPSLKS AREGSYPNQFDP (SEQ (SEQ ID NO: 35) (SEQ ID NO: 36)ID NO: 37) 22208 GSIKSGSHYWG NIYYSGSTYYNPSLKS AREGSYPNWFDP (SEQ(SEQ ID NO: 45) (SEQ ID NO: 46) ID NO: 47) 15462 GSISSSSYYWGNIYYSGSTYYNPSLKS AREGSYTTVLNV (SEQ (SEQ ID NO: 55) (SEQ ID NO: 56)ID NO: 57) 22213 GSIGRGSYYWG NIYYSGSTYYNPSLKS AREGSYTTVLNV (SEQ(SEO ID NO: 65) (SEO ID NO: 66) ID NO: 67) 15465 GSISSGGYYWSNIYYSGSTYYNPSLKS ARESSTISADFDL (SEQ (SEQ ID NO: 75) (SEQ ID NO: 76)ID NO: 77) 20506 GSISHGGYYWS NIYYSGSTYYNPSLKS ARESSTISADFDL (SEQ(SEQ ID NO: 85) (SEQ ID NO: 86) ID NO: 87) 15483 GSISSGGYYWSNIYYSGSTYYNPSLKS ARGLSTIDEAFDP (SEQ (SEQ ID NO: 95) (SEQ ID NO: 96)ID NO: 97) 20513 GSISDGSYYWS NIYYSGSTYYNPSLRS ARGLSTIDEAFDP (SEQ(SEQ ID NO: 105) (SEQ ID NO: 106) ID NO: 107) 22216 GSISDGSYYWSNIYYSGSTYYNPSLRS ARGLSTIDEAFDP (SEQ (SEQ ID NO: 115) (SEQ ID NO: 116)ID NO: 117) 15489 GSISSYYWS (SEQ YIYSSGSTNYNPSLKS ARGSGQYAAPDYGMDID NO: 125) (SEQ ID NO: 126) V (SEQ ID NO: 127) 20516 GSIISYYWG (SEQYIYSSGSTSYNPSLKS ARGSGLYAAPDYGLDV ID NO: 135) (SEQ ID NO: 136)(SEQ ID NO: 137) 15472 FTFSSYAMS (SEQ TISGSGGSTYYADSVKG ARGAGHYDLVGRYID NO: 145) (SEQ ID NO: 146) (SEQ ID NO: 147) 15503 FTFSSYAMS (SEQAISGSGGSTYYADSVKG ARVGFRALNY (SEQ ID ID NO: 155) (SEQ ID NO: 156)NO: 157) 15495 GTFSSYAIS (SEQ ID GIIPIFGTASYAQKFQG ARQQYDGRRYFGLNO: 165) (SEQ ID NO: 166) (SEQ ID NO: 167) 15478 GTFSSYAIS (SEQ IDGIIPIFGTANYAQKFQG ARGGPWFDP (SEQ ID NO: 175) (SEQ ID NO: 176) NO: 177)15441 FTFSSYAMS (SEQ AISGSGGSTSYADSVKG AKPSLATMLAFDI (SEQ ID NO: 185)(SEQ ID NO: 186) ID NO: 187) 20496 GSISSSVYYWS SILVSGSTYYNPSLKSARAVSFLDV (SEQ ID (SEQ ID NO: 195) (SEQ ID NO: 196) NO: 197) ¹The VHCDRs in Table 1 are determined according to Kabat.

TABLE 2 VL CDR Amino Acid Sequences² VL CDR1 VL CDR2 VL CDR3 Antibody(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 15461 RASQSVSSNLA GASTRAT (SEQ IDQQYHSFPFT (SEQ ID (SEQ ID NO: 8) NO: 9) NO: 10) 20500 RASQSVSSNLAGASTRAT (SEQ ID QQYHSFPFT (SEQ ID (SEQ ID NO: 18) NO: 19) NO: 20) 20501RASQSVSSNLA GASTRAT (SEQ ID QQYHSFPFT (SEQ ID (SEQ ID NO: 28) NO: 29)NO: 30) 20502 RASQSVSSNLA GASTRAT (SEQ ID QQYHSFPFT (SEQ ID(SEQ ID NO: 461) NO: 462) NO: 463) 20502.1 RASQSVSSNLA GASTRAT (SEQ IDQQYHSFPFT (SEQ ID (SEQ ID NO: 38) NO: 39) NO: 40) 22208 RASQSVSTNLADASARVT (SEQ ID QQYHSFPFT (SEQ ID (SEQ ID NO: 48) NO: 49) NO: 50) 15462RASQSVSSSYLA GASSRAT (SEQ ID QQAASYPLT (SEQ ID (SEQ ID NO: 58) NO: 59)NO: 60) 22213 RASQSVASSHLA DAVSRAT (SEQ ID QQAASYPLT (SEQ ID(SEQ ID NO: 68) NO: 69) NO: 70) 15465 RASQGISRWLA AASSLQS (SEQ IDQQAHTFPYT (SEQ ID (SEQ ID NO: 78) NO: 79) NO: 80) 20506 RASQGISRWLAAASSLQS (SEQ ID QQAHTFPYT (SEQ ID (SEQ ID NO: 88) NO: 89) NO: 90) 15483RASQSISSWLA KASSLES (SEQ ID QQDNSYPYT (SEQ ID (SEQ ID NO: 98) NO: 99)NO: 100) 20513 RASQSISSWLA KASSLES (SEQ ID QQDNSYPYT (SEQ ID(SEQ ID NO: 108) NO: 109) NO: 110) 22216 RASKSISSWLA EASSLHS (SEQ IDQQDNSYPYT (SEQ ID (SEQ ID NO: 118) NO: 119) NO: 120) 15489 RASQSISSWLAKASSLES (SEQ ID QQDNSFPFT (SEQ ID (SEQ ID NO: 128) NO: 129) NO: 130)20516 RASQSISSWLA KASSLES (SEQ ID QQDNSFPFT (SEQ ID (SEQ ID NO: 138)NO: 139) NO: 140) 15472 RASQSISSYLN AASSLQS (SEQ ID QQLYSLPPT (SEQ ID(SEQ ID NO: 148) NO: 149) NO: 150) 15503 RASQDISSWLA AASSLQS (SEQ IDQQATSYPPWT (SEQ ID (SEQ ID NO: 158) NO: 159) NO: 160) 15495 RASQSVSSNLASASTRAT (SEQ ID QQVNVWPPT (SEQ ID (SEQ ID NO: 168) NO: 169) NO: 170)15478 RASQSISSWLA KASSLES (SEQ ID QQYNSYPPFT (SEQ ID (SEQ ID NO: 178)NO: 179) NO: 180) 15441 RASQSISSWLA DASSLES (SEQ ID QQSKSYPRT (SEQ ID(SEQ ID NO: 188) NO: 189) NO: 190) 20496 RASQSISSYLN GASSLQS (SEQ ID NO:QQSYDPPWT (SEQ ID (SEQ ID NO: 198) 199) NO: 200) ²The VL CDRs in Table 2are determined according to Kabat.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the VH of anantibody listed in Table 3.

TABLE 3 Variable Heavy Chain (VH) Amino Acid Sequences AntibodyVH Amino Acid Sequence (SEQ ID NO) 15461QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNWFDPWGQGTLVTVSS (SEQ ID NO: 11) 20500QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSHYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLRSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNWFDPWGQGTLVTVSS (SEQ ID NO: 21) 20501QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSHYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNWLDPWGQGTLVTVSS (SEQ ID NO: 31) 20502QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLRSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNQFDPWGQGTLVTVSS (SEQ ID NO: 464) 20502.1QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNQFDPWGQGILVTVSS (SEQ ID NO: 41) 22208QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSHYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNWFDPWGQGTLVTVSS (SEQ ID NO: 51) 15462QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYTTVLNVWGQGTMVTVSS (SEQ ID NO: 61) 22213QLQLQESGPGLVKPSETLSLTCTVSGGSIGRGSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYTTVLNVWGQGTMVTVSS (SEQ ID NO: 71) 15465QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARESSTISADFDLWGRGTLVTVSS (SEQ ID NO: 81) 20506QLQLQESGPGLVKPSETLSLTCTASGGSISHGGYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARESSTISADFDLWGRGTLVTVSS (SEQ ID NO: 91) 15483QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGLSTIDEAFDPWGQGTLVTVSS (SEQ ID NO: 101) 20513QLQLQESGPGLVKPSETLSLTCTVSGGSISDGSYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGLSTIDEAFDPWGQGTLVTVSS (SEQ ID NO: 111) 22216QVQLQESGPGLVKPSQTLSLTCTVSGGSISDGSYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGLSTIDEAFDPWGQGTLVTVSS (SEQ ID NO: 121) 15489QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYSSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSGQYAAPDYGMDVWGQGTTVTVSS (SEQ ID NO: 131) 20516QVQLQESGPGLVKPSETLSLTCTVSGGSIISYYWGWIRQPPGKGLEWIGYIYSSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSGLYAAPDYGLDVWGQGTTVTVSS (SEQ ID NO: 141) 15472EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGAGHYDLVGRYWGQGTLVTVSS (SEQ ID NO: 151) 15503EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGFRALNYWGQGTTVTVSS (SEQ ID NO: 161) 15495QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTASYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARQQYDGRRYFGLWGRGTLVTVSS (SEQ ID NO: 171) 15478QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGPWFDPWGQGTLVTVSS (SEQ ID NO: 181) 15441EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPSLATMLAFDIWGQGTMVTVSS (SEQ ID NO: 191) 20496QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGSILVSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAVSFLDVWGQGTMVIVSS (SEQ ID NO: 201)

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the VL of anantibody listed in Table 4.

TABLE 4 Variable Light Chain (VL) Amino Acid Sequences AntibodyVL Amino Acid Sequence (SEQ ID NO) 15461EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIK (SEQ ID NO: 12) 20500EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIK (SEQ ID NO: 22) 20501EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIK (SEQ ID NO: 32) 20502 andEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLI 20502.1YGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIK (SEQ ID NO: 42) 22208EIVMTQSPATLSVSPGERATLSCRASQSVSTNLAWYQQKPGQAPRLLIYDASARVTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIK (SEQ ID NO: 52) 15462EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAASYPLTFGGGTKVEIK (SEQ ID NO: 62) 22213EIVLTQSPGTLSLSPGERATLSCRASQSVASSHLAWYQQKPGQAPRLLIYDAVSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAASYPLTFGGGTKVEIK (SEQ ID NO: 72) 15465DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHTFPYTFGGGTKVEIK (SEQ ID NO: 82) 20506DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHTFPYTFGGGTKVEIK (SEQ ID NO: 92) 15483DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSYPYTFGGGTKVEIK (SEQ ID NO: 102) 20513DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSYPYTFGGGTKVEIK (SEQ ID NO: 112) 22216DIQMTQSPSTLSASVGDRVTITCRASKSISSWLAWYQQKPGKAPKLLIYEASSLHSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSYPYTFGGGTKVEIK (SEQ ID NO: 122) 15489DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSFPFTFGGGTKVEIK (SEQ ID NO: 132) 20516DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSFPFTFGGGTKVEIK (SEQ ID NO: 142) 15472DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLYSLPPTFGGGTKVEIK (SEQ ID NO: 152) 15503DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQATSYPPWTFGGGTKVEIK (SEQ ID NO: 162) 15495EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYSASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQVNVWPPTFGGGTKVEIK (SEQ ID NO: 172) 15478DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPPFTFGGGTKVEIK (SEQ ID NO: 182) 15441DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSKSYPRTFGGGTKVEIK (SEQ ID NO: 192) 20496DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYDPPWTFGGGTKVEIK (SEQ ID NO: 202)

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the VH and the VL ofan antibody listed in Tables 3 and 4 (i.e., the VH of the antibodylisted in Table 3 and the VL of the same antibody listed in Table 4).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the VH frameworkregions of an antibody listed in Table 5.

TABLE 5 VH FR Amino Acid Sequences⁴ VH FR1 VH FR2 VH FR3 VH FR4 Antibody(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 15461 QLQLQESGPGLVKWIRQPPGKGLE RVTISVDTSKNQFSL WGQGTLVT PSETLSLTCTVSG WIG (SEQ IDKLSSVTAADTAVYY VSS (SEQ ID (SEQ ID NO: 205) NO: 206) C (SEQ ID NO: 207)NO: 208) 20500 QLQLQESGPGLVK WIRQPPGKGLE RVTISVDTSKNQFSL WGQGTLVTPSETLSLTCTVSG WIG (SEQ ID KLSSVTAADTAVYY VSS (SEQ ID (SEQ ID NO: 215)NO: 216) C (SEQ ID NO: 217) NO: 218) 20501 QLQLQESGPGLVK WIRQPPGKGLERVTISVDTSKNQFSL WGQGTLVT PSETLSLTCTVSG WIG (SEQ ID KLSSVTAADTAVYYVSS (SEQ ID (SEQ ID NO: 225) NO: 226) C (SEQ ID NO: 227) NO: 228) 20502QLQLQESGPGLVK WIRQPPGKGLE RVTISVDTSKNQFSL WGQGTLVT PSETLSLTCTVSGWIG (SEQ ID KLSSVTAADTAVYY VSS (SEQ ID (SEQ ID NO: 465) NO: 466)C (SEQ ID NO: 467) NO: 468) 20502.1 QLQLQESGPGLVK WIRQPPGKGLERVTISVDTSKNQFSL WGQGILVTV PSETLSLTCTVSG WIG (SEQ ID KLSSVTAADTAVYYSS (SEQ ID (SEQ ID NO: 235) NO: 236) C (SEQ ID NO: 237) NO: 238) 22208QLQLQESGPGLVK WIRQPPGKGLE RVTMSVDTSKNQFS WGQGTLVT PSETLSLTCTVSGWIG (SEQ ID LKLSSVTAADTAVY VSS (SEQ ID (SEQ ID NO: 245) NO: 246)YC (SEQ ID NO: 247) NO: 248) 15462 QLQLQESGPGLVK WIRQPPGKGLERVTISVDTSKNQFSL WGQGTMVT PSETLSLTCTVSG WIG (SEQ ID KLSSVTAADTAVYYVSS (SEQ ID (SEQ ID NO: 255) NO: 256) C (SEQ ID NO: 257) NO: 258) 22213QLQLQESGPGLVK WIRQPPGKGLE RVTISVDTSKNQFSL WGQGTMVT PSETLSLTCTVSGWIG (SEQ ID KLSSVTAADTAVYY VSS (SEQ ID (SEQ ID NO: 265) NO: 266)C (SEQ ID NO: 267) NO: 268) 15465 QVQLQESGPGLVK WIRQHPGKGLERVTISVDTSKNQFSL WGRGTLVT PSQTLSLTCTVSG WIG (SEQ ID KLSSVTAADTAVYYVSS (SEQ ID (SEQ ID NO: 275) NO: 276) C (SEQ ID NO: 277) NO: 278) 20506QLQLQESGPGLVK WIRQHPGKGLE RVTMSVDTSKNQFS WGRGTLVT PSETLSLTCTASGWIG (SEQ ID LKLSSVTAADTAVY VSS (SEQ ID (SEQ ID NO: 285) NO: 286)YC (SEQ ID NO: 287) NO: 288) 15483 QVQLQESGPGLVK WIRQHPGKGLERVTISVDTSKNQFSL WGQGTLVT PSQTLSLTCTVSG WIG (SEQ ID KLSSVTAADTAVYYVSS (SEQ ID (SEQ ID NO: 295) NO: 296) C (SEQ ID NO: 297) NO: 298) 20513QLQLQESGPGLVK WIRQHPGKGLE RVTMSVDTSKNQFS WGQGTLVT PSETLSLTCTVSGWIG (SEQ ID LKLSSVTAADTAVY VSS (SEQ ID (SEQ ID NO: 305) NO: 306)YC (SEQ ID NO: 307) NO: 308) 22216 QVQLQESGPGLVK WIRQHPGKGLERVTMSVDTSKNQFS WGQGTLVT PSQTLSLTCTVSG WIG (SEQ ID LKLSSVTAADTAVYVSS (SEQ ID (SEQ ID NO: 315) NO: 316) YC (SEQ ID NO: 317) NO: 318) 15489QVQLQESGPGLVK WIRQPPGKGLE RVTISVDTSKNQFSL WGQGTTVT PSETLSLTCTVSGWIG (SEQ ID KLSSVTAADTAVYY VSS (SEQ ID (SEQ ID NO: 325) NO: 326)C (SEQ ID NO: 327) NO: 328) 20516 QVQLQESGPGLVK WIRQPPGKGLERVTISVDTSKNQFSL WGQGTTVT PSETLSLTCTVSG WIG (SEQ ID KLSSVTAADTAVYYVSS (SEQ ID (SEQ ID NO: 335) NO: 336) C (SEQ ID NO: 337) NO: 338) 15472EVQLLESGGGLVQ WVRQAPGKGL RFTISRDNSKNTLYL WGQGTLVT PGGSLRLSCAASGEWVS (SEQ ID QMNSLRAEDTAVY VSS (SEQ ID (SEQ ID NO: 345) NO: 346)YC (SEQ ID NO: 347) NO: 348) 15503 EVQLLESGGGLVQ WVRQAPGKGLRFTISRDNSKNTLYL WGQGTTVT PGGSLRLSCAASG EWVS (SEQ ID QMNSLRAEDTAVYVSS (SEQ ID (SEQ ID NO: 355) NO: 356) YC (SEQ ID NO: 357) NO: 358) 15495QVQLVQSGAEVK WVRQAPGQGL RVTITADESTSTAY WGRGTLVT KPGSSVKVSCKASEWMG (SEQ ID MELSSLRSEDTAVY VSS (SEQ ID G (SEQ ID NO: 365) NO: 366)YC (SEQ ID NO: 367) NO: 368) 15478 QVQLVQSGAEVK WVRQAPGQGLRVTITADESTSTAY WGQGTLVT KPGSSVKVSCKAS EWMG (SEQ ID MELSSLRSEDTAVYVSS (SEQ ID G (SEQ ID NO: 375) NO: 376) YC (SEQ ID NO: 377) NO: 378)15441 EVQLLESGGGLVQ WVRQAPGKGL RFTISRDNSKNTLYL WGQGTMVT PGGSLRLSCAASGEWVS (SEQ ID QMNSLRAEDTAVY VSS (SEQ ID (SEQ ID NO: 385) NO: 386)YC (SEQ ID NO: 387) NO: 388) 20496 QLQLQESGPGLVK WIRQPPGKGLERVTISVDTSKNQFSL WGQGTMVI PSETLSLTCTVSG WIG (SEQ ID KLSSVTAADTAVYYVSS (SEQ ID (SEQ ID NO: 395) NO: 396) C (SEQ ID NO: 397) NO: 398) ⁴TheVH framework regions described in Table 5 are determined based upon theboundaries of the Kabat numbering system for CDRs. In other words, theVH CDRs are determined by Kabat and the framework regions are the aminoacid residues surrounding the CDRs in the variable region in the formatFR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the VL frameworkregions of an antibody listed in Table 6.

TABLE 6 VL FR Amino Acid Sequences³ VL FR1 VL FR2 VL FR3 VL FR4Anti-body (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 15461EIVMTQSPATLSVS WYQQKPGQAP GIPARFSGSGSGT FGGGTKVEI PGERATLSC (SEQRLLIY (SEQ ID EFTLTISSLQSEDF K (SEQ ID ID NO: 209) NO: 210)AVYYC (SEQ ID NO: 212) NO: 211) 20500 EIVMTQSPATLSVS WYQQKPGQAPGIPARFSGSGSGT FGGGTKVEI PGERATLSC (SEQ RLLIY (SEQ ID EFTLTISSLQSEDFK (SEQ ID ID NO: 219) NO: 220) AVYYC (SEQ ID NO: 222) NO: 221) 20501EIVMTQSPATLSVS WYQQKPGQAP GIPARFSGSGSGT FGGGTKVEI PGERATLSC (SEQRLLIY (SEQ ID EFTLTISSLQSEDF K (SEQ ID ID NO: 229) NO: 230)AVYYC (SEQ ID NO: 232) NO: 231) 20502 and EIVMTQSPATLSVS WYQQKPGQAPGIPARFSGSGSGT FGGGTKVEI 20502.1 PGERATLSC (SEQ RLLIY (SEQ IDEFTLTISSLQSEDF K (SEQ ID ID NO: 239) NO: 240) AVYYC (SEQ ID NO: 242)NO: 241) 22208 EIVMTQSPATLSVS WYQQKPGQAP GIPARFSGSGSGT FGGGTKVEIPGERATLSC (SEQ RLLIY (SEQ ID EFTLTISSLQSEDF K (SEQ ID ID NO: 249)NO: 250) AVYYC (SEQ ID NO: 252) NO: 251) 15462 EIVLTQSPGTLSLS WYQQKPGQAPGIPDRFSGSGSGT FGGGTKVEI PGERATLSC (SEQ RLLIY (SEQ ID DFTLTISRLEPEDFK (SEQ ID ID NO: 259) NO: 260) AVYYC (SEQ ID NO: 262) NO: 261) 22213EIVLTQSPGTLSLS WYQQKPGQAP GIPDRFSGSGSGT FGGGTKVEI PGERATLSC (SEQRLLIY (SEQ ID DFTLTISRLEPEDF K (SEQ ID ID NO: 269) NO: 270)AVYYC (SEQ ID NO: 272) NO: 271) 15465 DIQMTQSPSSVSAS WYQQKPGKAPGVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQ KLLIY (SEQ ID DFTLTISSLQPEDFK (SEQ ID ID NO: 279) NO: 280) ATYYC (SEQ ID NO: 282) NO: 281) 20506DIQMTQSPSSVSAS WYQQKPGKAP GVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQKLLIY (SEQ ID DFTLTISSLQPEDF K (SEQ ID ID NO: 289) NO: 290)ATYYC (SEQ ID NO: 292) NO: 291) 15483 DIQMTQSPSTLSAS WYQQKPGKAPGVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQ KLLIY (SEQ ID EFTLTISSLQPDDFK (SEQ ID ID NO: 299) NO: 300) ATYYC (SEQ ID NO: 302) NO: 301) 20513DIQMTQSPSTLSAS WYQQKPGKAP GVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQKLLIY (SEQ ID EFTLTISSLQPDDF K (SEQ ID ID NO: 309) NO: 310)ATYYC (SEQ ID NO: 312) NO: 311) 22216 DIQMTQSPSTLSAS WYQQKPGKAPGVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQ KLLIY (SEQ ID EFTLTISSLQPDDFK (SEQ ID ID NO: 319) NO: 320) ATYYC (SEQ ID NO: 322) NO: 321) 15489DIQMTQSPSTLSAS WYQQKPGKAP GVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQKLLIY (SEQ ID EFTLTISSLQPDDF K (SEQ ID ID NO: 329) NO: 330)ATYYC (SEQ ID NO: 332) NO: 331) 20516 DIQMTQSPSTLSAS WYQQKPGKAPGVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQ KLLIY (SEQ ID EFTLTISSLQPDDFK (SEQ ID ID NO: 339) NO: 340) ATYYC (SEQ ID NO: 342) NO: 341) 15472DIQMTQSPSSLSAS WYQQKPGKAP GVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQKLLIY (SEQ ID DFTLTISSLQPEDF K (SEQ ID ID NO: 349) NO: 350)ATYYC (SEQ ID NO: 352) NO: 351) 15503 DIQLTQSPSSVSAS WYQQKPGKAPGVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQ KLLIY (SEQ ID DFTLTISSLQPEDFK (SEQ ID ID NO: 359) NO: 360) ATYYC (SEQ ID NO: 362) NO: 361) 15495EIVMTQSPATLSVS WYQQKPGQAP GIPARFSGSGSGT FGGGTKVEI PGERATLSC (SEQRLLIY (SEQ ID EFTLTISSLQSEDF K (SEQ ID ID NO: 369) NO: 370)AVYYC (SEQ ID NO: 372) NO: 371) 15478 DIQMTQSPSTLSAS WYQQKPGKAPGVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQ KLLIY (SEQ ID EFTLTISSLQPDDFK (SEQ ID ID NO: 379) NO: 380) ATYYC (SEQ ID NO: 382) NO: 381) 15441DIQMTQSPSTLSAS WYQQKPGKAP GVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQKLLIY (SEQ ID EFTLTISSLQPDDF K (SEQ ID ID NO: 389) NO: 390)ATYYC (SEQ ID NO: 392) NO: 391) 20496 DIQMTQSPSSLSAS WYQQKPGKAPGVPSRFSGSGSGT FGGGTKVEI VGDRVTITC (SEQ KLLIY (SEQ ID DFTLTISSLQPEDFK (SEQ ID ID NO: 399) NO: 400) ATYYC (SEQ ID NO: 402) NO: 401) ³The VLframework regions described in Table 6 are determined based upon theboundaries of the Kabat numbering system for CDRs. In other words, theVL CDRs are determined by Kabat and the framework regions are the aminoacid residues surrounding the CDRs in the variable region in the formatFR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the four VHframework regions and the four VL framework regions of an antibodylisted in Tables 5 and 6 (i.e., the four VH framework regions of theantibody listed in Table 5 and the four VL framework regions of the sameantibody listed in Table 6.)

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the heavy chainsequence of an antibody listed in Table 7.

TABLE 7 Full-length heavy chain amino acid sequences AntibodyFull-Length Heavy Chain Amino Acid Sequence (SEQ ID NO) 15461QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13) 20500QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSHYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLRSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23) 20501QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSHYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNWLDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33) 20502QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLRSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNQFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 469) 20502.1QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNQFDPWGQGILVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 43) 22208QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSHYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53) 15462QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYTTVLNVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 63) 22213QLQLQESGPGLVKPSETLSLTCTVSGGSIGRGSYYWGWIRQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYTTVLNVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 73) 15465QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARESSTISADFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 83) 20506QLQLQESGPGLVKPSETLSLTCTASGGSISHGGYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARESSTISADFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93) 15483QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGLSTIDEAFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 103) 20513QLQLQESGPGLVKPSETLSLTCTVSGGSISDGSYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGLSTIDEAFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 113) 22216QVQLQESGPGLVKPSQTLSLTCTVSGGSISDGSYYWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGLSTIDEAFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 123) 15489QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYSSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSGQYAAPDYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 133) 20516QVQLQESGPGLVKPSETLSLTCTVSGGSIISYYWGWIRQPPGKGLEWIGYIYSSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSGLYAAPDYGLDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 143) 15472EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGAGHYDLVGRYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 153) 15503EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGFRALNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 163) 15495QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTASYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARQQYDGRRYFGLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 173) 15478QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGPWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 183) 15441EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPSLATMLAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 193) 20496QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGSILVSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAVSFLDVWGQGTMVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 203)

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the light chainsequence of an antibody listed in Table 8.

TABLE 8 Full-length light chain amino acid sequences AntibodyFull-Length Light Chain Amino Acid Sequence (SEQ ID NO) 15461EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14) 20500EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24) 20501EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 34) 20502 andEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLL 20502.1IYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 44) 22208EIVMTQSPATLSVSPGERATLSCRASQSVSTNLAWYQQKPGQAPRLLIYDASARVTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYHSFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 54) 15462EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAASYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 64) 22213EIVLTQSPGTLSLSPGERATLSCRASQSVASSHLAWYQQKPGQAPRLLIYDAVSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAASYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 74) 15465DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHTFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 84) 20506DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHTFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 94) 15483DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 104) 20513DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 114) 22216DIQMTQSPSTLSASVGDRVTITCRASKSISSWLAWYQQKPGKAPKLLIYEASSLHSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 124) 15489DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 134) 20516DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDNSFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 144) 15472DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLYSLPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 154) 15503DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQATSYPPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 164) 15495EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYSASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQVNVWPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 174) 15478DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 184) 15441DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSKSYPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 194) 20496DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYDPPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 204)

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4 and comprises the heavy chainsequence and the light chain sequence of an antibody listed in Tables 7and 8 (i.e., the heavy chain sequence of the antibody listed in Table 7and the light chain sequence of the same antibody listed in Table 8).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), and comprises a VH comprising a sequence at least80% identical to the VH sequence of the same antibody in Table 3 and aVL comprising a sequence at least 80% identical to the VL sequence ofthe same antibody in Table 4. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human B7-H4,comprises the six CDRs of an antibody listed in Tables 1 and 2 (i.e.,the three VH CDRs of the antibody listed in Table 1 and the three VLCDRs of the same antibody listed in Table 2), and comprises a VHcomprising a sequence at least 85% identical to the VH sequence of thesame antibody in Table 3 and a VL comprising a sequence at least 85%identical to the VL sequence of the same antibody in Table 4.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), and comprises a VH comprising a sequence at least90% identical to the VH sequence of the same antibody in Table 3 and aVL comprising a sequence at least 90% identical to the VL sequence ofthe same antibody in Table 4. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human B7-H4,comprises the six CDRs of an antibody listed in Tables 1 and 2 (i.e.,the three VH CDRs of the antibody listed in Table 1 and the three VLCDRs of the same antibody listed in Table 2), and comprises a VHcomprising a sequence at least 95% identical to the VH sequence of thesame antibody in Table 3 and a VL comprising a sequence at least 95%identical to the VL sequence of the same antibody in Table 4.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), and comprises a VH comprising a sequence at least96% identical to the VH sequence of the same antibody in Table 3 and aVL comprising a sequence at least 96% identical to the VL sequence ofthe same antibody in Table 4. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human B7-H4,comprises the six CDRs of an antibody listed in Tables 1 and 2 (i.e.,the three VH CDRs of the antibody listed in Table 1 and the three VLCDRs of the same antibody listed in Table 2), and comprises a VHcomprising a sequence at least 97% identical to the VH sequence of thesame antibody in Table 3 and a VL comprising a sequence at least 97%identical to the VL sequence of the same antibody in Table 4. In certainembodiments, an antibody or antigen-binding fragment thereof describedherein binds to human B7-H4, comprises the six CDRs of an antibodylisted in Tables 1 and 2 (i.e., the three VH CDRs of the antibody listedin Table 1 and the three VL CDRs of the same antibody listed in Table2), and comprises a VH comprising a sequence at least 98% identical tothe VH sequence of the same antibody in Table 3 and a VL comprising asequence at least 98% identical to the VL sequence of the same antibodyin Table 4. In certain embodiments, an antibody or antigen-bindingfragment thereof described herein binds to human B7-H4, comprises thesix CDRs of an antibody listed in Tables 1 and 2 (i.e., the three VHCDRs of the antibody listed in Table 1 and the three VL CDRs of the sameantibody listed in Table 2), and comprises a VH comprising a sequence atleast 99% identical to the VH sequence of the same antibody in Table 3and a VL comprising a sequence at least 99% identical to the VL sequenceof the same antibody in Table 4.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 80%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 80% identical to the VL sequence of thesame antibody in Table 4, and binds to human, cynomolgus monkey, rat,and/or mouse B7-H4. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human B7-H4,comprises the six CDRs of an antibody listed in Tables 1 and 2 (i.e.,the three VH CDRs of the antibody listed in Table 1 and the three VLCDRs of the same antibody listed in Table 2), comprises a VH comprisinga sequence at least 85% identical to the VH sequence of the sameantibody in Table 3 and a VL comprising a sequence at least 85%identical to the VL sequence of the same antibody in Table 4, and bindsto human, cynomolgus monkey, rat, and/or mouse B7-H4.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 90%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 90% identical to the VL sequence of thesame antibody in Table 4, and binds to human, cynomolgus monkey, rat,and/or mouse B7-H4. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human B7-H4,comprises the six CDRs of an antibody listed in Tables 1 and 2 (i.e.,the three VH CDRs of the antibody listed in Table 1 and the three VLCDRs of the same antibody listed in Table 2), comprises a VH comprisinga sequence at least 95% identical to the VH sequence of the sameantibody in Table 3 and a VL comprising a sequence at least 95%identical to the VL sequence of the same antibody in Table 4, and bindsto human, cynomolgus monkey, rat, and/or mouse B7-H4.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 96%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 96% identical to the VL sequence of thesame antibody in Table 4, and binds to human, cynomolgus monkey, rat,and/or mouse B7-H4. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human B7-H4,comprises the six CDRs of an antibody listed in Tables 1 and 2 (i.e.,the three VH CDRs of the antibody listed in Table 1 and the three VLCDRs of the same antibody listed in Table 2), comprises a VH comprisinga sequence at least 97% identical to the VH sequence of the sameantibody in Table 3 and a VL comprising a sequence at least 97%identical to the VL sequence of the same antibody in Table 4, and bindsto human, cynomolgus monkey, rat, and/or mouse B7-H4. In certainembodiments, an antibody or antigen-binding fragment thereof describedherein binds to human B7-H4, comprises the six CDRs of an antibodylisted in Tables 1 and 2 (i.e., the three VH CDRs of the antibody listedin Table 1 and the three VL CDRs of the same antibody listed in Table2), comprises a VH comprising a sequence at least 98% identical to theVH sequence of the same antibody in Table 3 and a VL comprising asequence at least 98% identical to the VL sequence of the same antibodyin Table 4, and binds to human, cynomolgus monkey, rat, and/or mouseB7-H4. In certain embodiments, an antibody or antigen-binding fragmentthereof described herein binds to human B7-H4, comprises the six CDRs ofan antibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 99%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 99% identical to the VL sequence of thesame antibody in Table 4, and binds to human, cynomolgus monkey, rat,and/or mouse B7-H4.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 80%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 80% identical to the VL sequence of thesame antibody in Table 4, increases T cell proliferation, increases IFNγproduction, and mediates ADCC activity against B7-H4-expressing cells.In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 85%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 85% identical to the VL sequence of thesame antibody in Table 4, increases T cell proliferation, increases IFNγproduction, and mediates ADCC activity against B7-H4-expressing cells.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 90%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 90% identical to the VL sequence of thesame antibody in Table 4, increases T cell proliferation, increases IFNγproduction, and/or mediates ADCC activity against B7-H4-expressingcells. In certain embodiments, an antibody or antigen-binding fragmentthereof described herein binds to human B7-H4, comprises the six CDRs ofan antibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 95%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 95% identical to the VL sequence of thesame antibody in Table 4, increases T cell proliferation, increases IFNγproduction, and mediates ADCC activity against B7-H4-expressing cells.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 96%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 96% identical to the VL sequence of thesame antibody in Table 4, increases T cell proliferation, increases IFNγproduction, and mediates ADCC activity against B7-H4-expressing cells.In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 97%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 97% identical to the VL sequence of thesame antibody in Table 4, increases T cell proliferation, increases IFNγproduction, and mediates ADCC activity against B7-H4-expressing cells.In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 98%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 98% identical to the VL sequence of thesame antibody in Table 4, increases T cell proliferation, increases IFNγproduction, and mediates ADCC activity against B7-H4-expressing cells.In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human B7-H4, comprises the six CDRs of anantibody listed in Tables 1 and 2 (i.e., the three VH CDRs of theantibody listed in Table 1 and the three VL CDRs of the same antibodylisted in Table 2), comprises a VH comprising a sequence at least 99%identical to the VH sequence of the same antibody in Table 3 and a VLcomprising a sequence at least 99% identical to the VL sequence of thesame antibody in Table 4, increases T cell proliferation, increases IFNγproduction, and mediates ADCC activity against B7-H4-expressing cells.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein may be described by its VL domain alone, or its VHdomain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See,for example, Rader C et al., (1998) PNAS 95: 8910-8915, which isincorporated herein by reference in its entirety, describing thehumanization of the mouse anti-αvβ3 antibody by identifying acomplementing light chain or heavy chain, respectively, from a humanlight chain or heavy chain library, resulting in humanized antibodyvariants having affinities as high or higher than the affinity of theoriginal antibody. See also Clackson T et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety,describing methods of producing antibodies that bind a specific antigenby using a specific VL domain (or VH domain) and screening a library forthe complementary variable domains. The screen produced 14 new partnersfor a specific VH domain and 13 new partners for a specific VL domain,which were strong binders, as determined by ELISA. See also Kim S J &Hong H J, (2007) J Microbiol 45: 572-577, which is incorporated hereinby reference in its entirety, describing methods of producing antibodiesthat bind a specific antigen by using a specific VH domain and screeninga library (e.g., human VL library) for complementary VL domains; theselected VL domains in turn could be used to guide selection ofadditional complementary (e.g., human) VH domains.

In certain aspects, the CDRs of an antibody or antigen-binding fragmentthereof can be determined according to the Chothia numbering scheme,which refers to the location of immunoglobulin structural loops (see,e.g., Chothia C & Lesk A M, (1987), J Mol Biol 196: 901-917; Al-LazikaniB et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J MolBiol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226). Typically, when using the Kabatnumbering convention, the Chothia CDR-H1 loop is present at heavy chainamino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present atheavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is presentat heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop ispresent at light chain amino acids 24 to 34, the Chothia CDR-L2 loop ispresent at light chain amino acids 50 to 56, and the Chothia CDR-L3 loopis present at light chain amino acids 89 to 97. The end of the ChothiaCDR-H1 loop when numbered using the Kabat numbering convention variesbetween H32 and H34 depending on the length of the loop (this is becausethe Kabat numbering scheme places the insertions at H35A and H35B; ifneither 35A nor 35B is present, the loop ends at 32; if only 35A ispresent, the loop ends at 33; if both 35A and 35B are present, the loopends at 34).

In certain aspects, provided herein are antibodies and antigen-bindingfragments thereof that specifically bind to B7-H4 (e.g., human B7-H4)and comprise the Chothia VH and VL CDRs of an antibody listed in Tables3 and 4. In certain embodiments, antibodies or antigen-binding fragmentsthereof that specifically bind to B7-H4 (e.g., human B7-H4) comprise oneor more CDRs, in which the Chothia and Kabat CDRs have the same aminoacid sequence. In certain embodiments, provided herein are antibodiesand antigen-binding fragments thereof that specifically bind to B7-H4(e.g., human B7-H4) and comprise combinations of Kabat CDRs and ChothiaCDRs.

In certain aspects, the CDRs of an antibody or antigen-binding fragmentthereof can be determined according to the IMGT numbering system asdescribed in Lefranc M-P, (1999) The Immunologist 7: 132-136 and LefrancM-P et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGTnumbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is atpositions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is atpositions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is atpositions 89 to 97. In a particular embodiment, provided herein areantibodies and antigen-binding fragments thereof that specifically bindto B7-H4 (e.g., human B7-H4) and comprise the IMGT VH and VL CDRs of anantibody listed in Tables 3 and 4, for example, as described in LefrancM-P (1999) supra and Lefranc M-P et al., (1999) supra).

In certain aspects, the CDRs of an antibody or antigen-binding fragmentthereof can be determined according to MacCallum R M et al., (1996) JMol Biol 262: 732-745. See also, e.g., Martin A. “Protein Sequence andStructure Analysis of Antibody Variable Domains,” in AntibodyEngineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439,Springer-Verlag, Berlin (2001). In a particular embodiment, providedherein are antibodies or antigen-binding fragments thereof thatspecifically bind to B7-H4 (e.g., human B7-H4) and comprise VH and VLCDRs of an antibody listed in Tables 3 and 4 as determined by the methodin MacCallum R M et al.

In certain aspects, the CDRs of an antibody or antigen-binding fragmentthereof can be determined according to the AbM numbering scheme, whichrefers AbM hypervariable regions which represent a compromise betweenthe Kabat CDRs and Chothia structural loops, and are used by OxfordMolecular's AbM antibody modeling software (Oxford Molecular Group,Inc.). In a particular embodiment, provided herein are antibodies orantigen-binding fragments thereof that specifically bind to B7-H4 (e.g.,human B7-H4) and comprise VH and VL CDRs of an antibody listed in Tables3 and 4 as determined by the AbM numbering scheme.

In specific aspects, provided herein are antibodies that comprise aheavy chain and a light chain. With respect to the heavy chain, in aspecific embodiment, the heavy chain of an antibody described herein canbe an alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavychain. In another specific embodiment, the heavy chain of an antibodydescribed can comprise a human alpha (α), delta (δ), epsilon (ε), gamma(γ) or mu (μ) heavy chain. In a particular embodiment, an antibodydescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4), comprises a heavy chain wherein the amino acid sequence of theVH domain comprises an amino acid sequence set forth in Table 3 andwherein the constant region of the heavy chain comprises the amino acidsequence of a human gamma (γ) heavy chain constant region. In a specificembodiment, an antibody described herein, which specifically binds toB7-H4 (e.g., human B7-H4), comprises a heavy chain wherein the aminoacid sequence of the VH domain comprises a sequence set forth in Table3, and wherein the constant region of the heavy chain comprises theamino acid of a human heavy chain described herein or known in the art.Non-limiting examples of human constant region sequences have beendescribed in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A etal., (1991) supra.

With respect to the light chain, in a specific embodiment, the lightchain of an antibody described herein is a kappa light chain. Theconstant region of a human kappa light chain can comprise the followingamino acid sequence:

(SEQ ID NO: 405) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.

The constant region of a human kappa light chain can be encoded by thefollowing nucleotide sequence:

(SEQ ID NO: 406) CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT.

In another specific embodiment, the light chain of an antibody describedherein is a lambda light chain. In yet another specific embodiment, thelight chain of an antibody described herein is a human kappa light chainor a human lambda light chain. In a particular embodiment, an antibodydescribed herein, which immunospecifically binds to a B7-H4 polypeptide(e.g., human B7-H4) comprises a light chain wherein the amino acidsequence of the VL domain comprises a sequence set forth in Table 4, andwherein the constant region of the light chain comprises the amino acidsequence of a human kappa light chain constant region. In anotherparticular embodiment, an antibody described herein, whichimmunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a lightchain wherein the amino acid sequence of the VL domain comprises asequence set forth in Table 4 and wherein the constant region of thelight chain comprises the amino acid sequence of a human lambda lightchain constant region. In a specific embodiment, an antibody describedherein, which immunospecifically binds to B7-H4 (e.g., human B7-H4)comprises a light chain wherein the amino acid sequence of the VL domaincomprises a sequence set forth in Table 4 and wherein the constantregion of the light chain comprises the amino acid sequence of a humankappa or lambda light chain constant region. Non-limiting examples ofhuman constant region sequences have been described in the art, e.g.,see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra.

In a specific embodiment, an antibody described herein, whichimmunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VHdomain and a VL domain comprising any amino acid sequence describedherein, and wherein the constant regions comprise the amino acidsequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgYimmunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgYimmunoglobulin molecule. In another specific embodiment, an antibodydescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4) comprises a VH domain and a VL domain comprising any amino acidsequence described herein, and wherein the constant regions comprise theamino acid sequences of the constant regions of an IgG, IgE, IgM, IgD,IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) ofimmunoglobulin molecule. In a particular embodiment, the constantregions comprise the amino acid sequences of the constant regions of ahuman IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g.,IgG2a and IgG2b) of immunoglobulin molecule.

The constant region of a human IgG1 heavy chain can comprise thefollowing amino acid sequence:

(SEQ ID NO: 407) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The constant region of a human IgG₁ heavy chain can be encoded by thefollowing nucleotide sequence:

(SEQ ID NO: 408) GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA.

Non-limiting examples of human constant regions are described in theart, e.g., see Kabat E A et al., (1991) supra.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibody orantigen-binding fragment thereof described herein (e.g., CH2 domain(residues 231-340 of human IgG₁) and/or CH3 domain (residues 341-447 ofhuman IgG₁) and/or the hinge region, with numbering according to theKabat numbering system (e.g., the EU index in Kabat)) to alter one ormore functional properties of the antibody or antigen-binding fragmentthereof, such as serum half-life, complement fixation, Fc receptorbinding, and/or antigen-dependent cellular cytotoxicity.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain may be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody or antigen-binding fragment thereof.

In some embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibody orantigen-binding fragment thereof described herein (e.g., CH2 domain(residues 231-340 of human IgG1) and/or CH3 domain (residues 341-447 ofhuman IgG1) and/or the hinge region, with numbering according to theKabat numbering system (e.g., the EU index in Kabat)) to increase ordecrease the affinity of the antibody or antigen-binding fragmentthereof for an Fc receptor (e.g., an activated Fc receptor) on thesurface of an effector cell. Mutations in the Fc region that decrease orincrease affinity for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor that can bemade to alter the affinity of the antibody or antigen-binding fragmentthereof for an Fc receptor are described in, e.g., Smith P et al.,(2012) PNAS 109: 6181-6186, U.S. Pat. No. 6,737,056, and InternationalPublication Nos. WO 02/060919; WO 98/23289; and WO 97/34631, which areincorporated herein by reference.

In a specific embodiment, one, two, or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody or antigen-binding fragment thereof in vivo.See, e.g., International Publication Nos. WO 02/060919; WO 98/23289; andWO 97/34631; and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375 and6,165,745 for examples of mutations that will alter (e.g., decrease orincrease) the half-life of an antibody or antigen-binding fragmentthereof in vivo. In some embodiments, one, two or more amino acidmutations (i.e., substitutions, insertions, or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to decrease the half-lifeof the antibody or antigen-binding fragment thereof in vivo. In otherembodiments, one, two or more amino acid mutations (i.e., substitutions,insertions or deletions) are introduced into an IgG constant domain, orFcRn-binding fragment thereof (preferably an Fc or hinge-Fc domainfragment) to increase the half-life of the antibody or antigen-bindingfragment thereof in vivo. In a specific embodiment, the antibodies orantigen-binding fragments thereof may have one or more amino acidmutations (e.g., substitutions) in the second constant (CH2) domain(residues 231-340 of human IgG1) and/or the third constant (CH3) domain(residues 341-447 of human IgG1), with numbering according to the EUindex in Kabat (Kabat E A et al., (1991) supra). In a specificembodiment, the constant region of the IgG1 comprises a methionine (M)to tyrosine (Y) substitution in position 252, a serine (S) to threonine(T) substitution in position 254, and a threonine (T) to glutamic acid(E) substitution in position 256, numbered according to the EU index asin Kabat. See U.S. Pat. No. 7,658,921, which is incorporated herein byreference. This type of mutant IgG, referred to as “YTE mutant” has beenshown to display fourfold increased half-life as compared to wild-typeversions of the same antibody (see Dall'Acqua W F et al., (2006) J BiolChem 281: 23514-24). In certain embodiments, an antibody orantigen-binding fragment thereof comprises an IgG constant domaincomprising one, two, three or more amino acid substitutions of aminoacid residues at positions 251-257, 285-290, 308-314, 385-389, and428-436, numbered according to the EU index as in Kabat.

In a further embodiment, one, two, or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the antibody or antigen-binding fragment thereof. Forexample, one or more amino acids selected from amino acid residues 234,235, 236, 237, 297, 318, 320 and 322, numbered according to the EU indexas in Kabat, can be replaced with a different amino acid residue suchthat the antibody or antigen-binding fragment thereof has an alteredaffinity for an effector ligand but retains the antigen-binding abilityof the parent antibody. The effector ligand to which affinity is alteredcan be, for example, an Fc receptor or the C1 component of complement.This approach is described in further detail in U.S. Pat. Nos. 5,624,821and 5,648,260. In some embodiments, the deletion or inactivation(through point mutations or other means) of a constant region domain mayreduce Fc receptor binding of the circulating antibody orantigen-binding fragment thereof thereby increasing tumor localization.See, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886 for a description ofmutations that delete or inactivate the constant domain and therebyincrease tumor localization. In certain embodiments, one or more aminoacid substitutions can be introduced into the Fc region to removepotential glycosylation sites on Fc region, which may reduce Fc receptorbinding (see, e.g., Shields R L et al., (2001) J Biol Chem 276:6591-604).

In certain embodiments, one or more amino acids selected from amino acidresidues 329, 331, and 322 in the constant region, numbered according tothe EU index as in Kabat, can be replaced with a different amino acidresidue such that the antibody or antigen-binding fragment thereof hasaltered C1q binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al). In some embodiments, one or moreamino acid residues within amino acid positions 231 to 238 in theN-terminal region of the CH2 domain are altered to thereby alter theability of the antibody to fix complement. This approach is describedfurther in International Publication No. WO 94/29351. In certainembodiments, the Fc region is modified to increase the ability of theantibody or antigen-binding fragment thereof to mediate antibodydependent cellular cytotoxicity (ADCC) and/or to increase the affinityof the antibody or antigen-binding fragment thereof for an Fey receptorby mutating one or more amino acids (e.g., introducing amino acidsubstitutions) at the following positions: 238, 239, 248, 249, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309,312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335,337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419,430, 434, 435, 437, 438, or 439, numbered according to the EU index asin Kabat. This approach is described further in InternationalPublication No. WO 00/42072.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein comprises the constant domain of an IgG1 with amutation (e.g., substitution) at position 267, 328, or a combinationthereof, numbered according to the EU index as in Kabat. In certainembodiments, an antibody or antigen-binding fragment thereof describedherein comprises the constant domain of an IgG1 with a mutation (e.g.,substitution) selected from the group consisting of S267E, L328F, and acombination thereof. In certain embodiments, an antibody orantigen-binding fragment thereof described herein comprises the constantdomain of an IgG1 with a S267E/L328F mutation (e.g., substitution). Incertain embodiments, an antibody or antigen-binding fragment thereofdescribed herein comprising the constant domain of an IgG1 with aS267E/L328F mutation (e.g., substitution) has an increased bindingaffinity for FcγRIIA, FcγRIIB, or FcγRIIA and FcγRIIB.

Antibodies with reduced fucose content have been reported to have anincreased affinity for Fc receptors, such as, e.g., FcγRIIIAAccordingly, in certain embodiments, an antibody or antigen-bindingfragment thereof described herein has reduced fucose content or lacksfucose (i.e., is “afucosylated”). Such antibodies or antigen-bindingfragments thereof can be produced using techniques known to one skilledin the art. For example, they can be expressed in cells deficient orlacking the ability to fucosylate. In a specific example, cell lineswith a knockout of both alleles of α1,6-fucosyltransferase can be usedto produce antibodies or antigen-binding fragments thereof with reducedfucose content. The Potelligent® system (Lonza) is an example of such asystem that can be used to produce antibodies and antigen-bindingfragments thereof with reduced fucose content. Alternatively, antibodiesor antigen-binding fragments thereof with reduced fucose content or nofucose content can be produced by, e.g.: (i) culturing cells underconditions which prevent or reduce fucosylation; (ii) posttranslationalremoval of fucose (e.g., with a fucosidase enzyme); (iii)post-translational addition of the desired carbohydrate, e.g., afterrecombinant expression of a non-glycosylated glycoprotein; or (iv)purification of the glycoprotein so as to select for antibodies orantigen-binding fragments thereof which are not fucsoylated. See, e.g.,Longmore G D & Schachter H (1982) Carbohydr Res 100: 365-92 andImai-Nishiya H et al., (2007) BMC Biotechnol. 7: 84 for methods forproducing antibodies thereof with no fucose content or reduced fucosecontent. See also Example 8 herein describing the production ofafucosylated B7-H4 antibodies.

In some embodiments, the B7-H4 antibody or antigen-binding fragmentthereof has enhanced ADCC activity in vitro compared to fucosylatedB7-H4 antibodies or antigen-binding fragments thereof having the sameamino acid sequence. In some embodiments, the afucosylated B7-H4antibodies or antigen-binding fragments thereof cause specific lysisthat is at least 10, at least 15, at least 20, at least 25, at least 30,at least 35, at least 40, at least 45, at least 50, at least 60, atleast 65, at least 70, or at least 75 percentage points greater thanspecific lysis with fucosylated B7-H4 antibodies.

In some embodiments, the B7-H4 antibody or antigen-binding fragmentthereof has enhanced affinity for Fc gamma RIIIA compared to fucosylatedB7-H4 antibodies or antigen-binding fragments thereof having the sameamino acid sequence. In some embodiments, the afucosylated B7-H4antibodies or antigen-binding fragments thereof bind to Fc gamma RIIIAwith at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold,at least 7-fold, at least 10-fold, at least 12-fold, at least 15-fold,at least 17-fold, or at least 20-fold greater affinity than fucosylatedB7-H4 antibodies or antigen-binding fragments thereof. In someembodiments, affinity for Fc gamma RIIIA is determined using surfaceplasmon resonance. In some embodiments, Fc gamma RIIIA is selected fromFc gamma RIIIA(V158) and Fc gamma RIIIA(F158). In some embodiments, Fcgamma RIIIA is Fc gamma RIIIA(V158).

In some embodiments, the presence of fucose can be determined by amethod comprising high performance liquid chromatography (HPLC),capillary electrophoresis, or MALDI-TOF mass spectrometry.

In specific embodiments, an antibody or antigen-binding fragment thereof(i) comprises the CDR sequences of 20502 (e.g., the amino acid sequencesof SEQ ID NOs:458-463), the VH and VL sequences of 20502 (the amino acidsequences of SEQ ID NOs:464 and 42, respectively), or the heavy andlight chain sequences of 20502 (the amino acid sequences of SEQ IDNOs:469 and 44, respectively) and (ii) is afucosylated.

In specific embodiments, a composition comprises antibodies orantigen-binding fragments thereof that (i) comprises the CDR sequencesof 20502 (e.g., the amino acid sequences of SEQ ID NOs:458-463), the VHand VL sequences of 20502 (the amino acid sequences of SEQ ID NOs:464and 42, respectively), or the heavy and light chain sequences of 20502(the amino acid sequences of SEQ ID NOs:469 and 44, respectively) and(ii) are afucosylated, e.g., wherein at least 95% of the antibodies inthe composition are afucosylated or wherein fucosylation is undetectablein the composition.

In specific embodiments, an antibody or antigen-binding fragment thereof(i) comprises the CDR sequences of 20502.1 (e.g., the amino acidsequences of SEQ ID NOs:35-40), the VH and VL sequences of 20502.1 (theamino acid sequences of SEQ ID NOs:41 and 42, respectively), or theheavy and light chain sequences of 20502.1 (the amino acid sequences ofSEQ ID NOs:43 and 44, respectively) and (ii) is afucosylated.

In specific embodiments, a composition comprises antibodies orantigen-binding fragments thereof that (i) comprises the CDR sequencesof 20502.1 (e.g., the amino acid sequences of SEQ ID NOs:35-40), the VHand VL sequences of 20502.1 (the amino acid sequences of SEQ ID NOs:41and 42, respectively), or the heavy and light chain sequences of 20502.1(the amino acid sequences of SEQ ID NOs:43 and 44, respectively) and(ii) are afucosylated, e.g., wherein at least 95% of the antibodies inthe composition are afucosylated or wherein fucosylation is undetectablein the composition.

In specific embodiments, an antibody or antigen-binding fragment thereof(i) comprises the CDR sequences of 22213 (e.g., the amino acid sequencesof SEQ ID NOs:65-70), the VH and VL sequences of 22213 (the amino acidsequences of SEQ ID NOs:71 and 72, respectively), or the heavy and lightchain sequences of 22213 (the amino acid sequences of SEQ ID NOs:73 and74, respectively) and (ii) is afucosylated.

In specific embodiments, a composition comprises antibodies orantigen-binding fragments thereof that (i) comprises the CDR sequencesof 22213 (e.g., the amino acid sequences of SEQ ID NOs:65-70), the VHand VL sequences of 22213 (the amino acid sequences of SEQ ID NOs:71 and72, respectively), or the heavy and light chain sequences of 22213 (theamino acid sequences of SEQ ID NOs:73 and 74, respectively) and (ii) areafucosylated, e.g., wherein at least 95% of the antibodies in thecomposition are afucosylated or wherein fucosylation is undetectable inthe composition.

Engineered glycoforms may be useful for a variety of purposes, includingbut not limited to enhancing or reducing effector function. Methods forgenerating engineered glycoforms in an antibody or antigen-bindingfragment thereof described herein include but are not limited to thosedisclosed, e.g., in Umalia P et al., (1999) Nat Biotechnol 17: 176-180;Davies J et al., (2001) Biotechnol Bioeng 74: 288-294; Shields R L etal., (2002) J Biol Chem 277: 26733-26740; Shinkawa T et al., (2003) JBiol Chem 278: 3466-3473; Niwa R et al., (2004) Clin Cancer Res 1:6248-6255; Presta L G et al., (2002) Biochem Soc Trans 30: 487-490;Kanda Y et al., (2007) Glycobiology 17: 104-118; U.S. Pat. Nos.6,602,684; 6,946,292; and 7,214,775; U.S. Patent Publication Nos. US2007/0248600; 2007/0178551; 2008/0060092; and 2006/0253928;International Publication Nos. WO 00/61739; WO 01/292246; WO 02/311140;and WO 02/30954; Potillegent™ technology (Biowa, Inc. Princeton, N.J.);and GlycoMAb® glycosylation engineering technology (Glycartbiotechnology AG, Zurich, Switzerland). See also, e.g., Ferrara C etal., (2006) Biotechnol Bioeng 93: 851-861; International PublicationNos. WO 07/039818; WO 12/130831; WO 99/054342; WO 03/011878; and WO04/065540.

In certain embodiments, any of the constant region mutations ormodifications described herein can be introduced into one or both heavychain constant regions of an antibody or antigen-binding fragmentthereof described herein having two heavy chain constant regions.

In another particular embodiment, an antibody or antigen-bindingfragment thereof described herein, which immunospecifically binds toB7-H4 (e.g., human B7-H4), comprises a heavy chain and a light chain,wherein (i) the heavy chain comprises a VH domain comprising the VHCDR1, VL CDR2, and VL CDR3 amino acid sequences of an antibody listed inTable 1 (e.g., SEQ ID NOs:458-460, 35-37, or 65-67); (ii) the lightchain comprises a VL domain comprising the VL CDR1, VH CDR2, and VH CDR3amino acid sequences of the same antibody listed in Table 2 (e.g., SEQID NOs:461-463, 38-40, or 68-70); (iii); and the heavy chain furthercomprises a constant heavy chain domain comprising the amino acidsequence of the constant domain of a human IgG1 heavy chain (iv); thelight chain further comprises a constant light chain domain comprisingthe amino acid sequence of the constant domain of a human kappa lightchain.

In another particular embodiment, an antibody or antigen-bindingfragment thereof described herein, which immunospecifically binds toB7-H4 (e.g., human B7-H4), comprises a heavy chain and a light chain,wherein (i) the heavy chain comprises a VH domain comprising the aminoacid sequence of an antibody listed in Table 3 (e.g., SEQ ID NO:464, 41,or 71); (ii) the light chain comprises a VL domain comprising the aminoacid sequence of the same antibody listed in Table 4 (e.g., SEQ ID NO:42or 73); (iii); and the heavy chain further comprises a constant heavychain domain comprising the amino acid sequence of the constant domainof a human IgG1 heavy chain (iv); the light chain further comprises aconstant light chain domain comprising the amino acid sequence of theconstant domain of a human kappa light chain.

In specific embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4) exhibits T cell checkpoint blockade activity. Exemplary methodsof measuring T cell checkpoint blockade activity are provided herein inExamples 7 and 11. In specific embodiments, an antibody orantigen-binding fragment thereof described herein, whichimmunospecifically binds to B7-H4 (e.g., human B7-H4) increasesinterferon-gamma (IFNγ) production in T cells. Exemplary methods ofmeasuring IFNγ production are provided herein in Examples 7 and 11. Inspecific embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4) increases T cell proliferation. Exemplary methods of measuringT-cell proliferation are provided herein in Example 7. In specificembodiments, an antibody or antigen-binding fragment thereof describedherein, which immunospecifically binds to B7-H4 (e.g., human B7-H4)increases CD4+ T cell proliferation. Exemplary methods of measuring CD4+T-cell proliferation are provided herein in Example 7. In specificembodiments, an antibody or antigen-binding fragment thereof describedherein, which immunospecifically binds to B7-H4 (e.g., human B7-H4)increases CD8+ T cell proliferation. Exemplary methods of measuring CD8+T-cell proliferation are provided herein in Example 7.

In specific embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4) exhibits antibody-dependent cellular cytotoxicity (ADCC)activity. In specific embodiments, an antibody or antigen-bindingfragment thereof described herein, which immunospecifically binds toB7-H4 (e.g., human B7-H4) exhibits antibody-dependent cellularcytotoxicity (ADCC) activity on cell lines with at least 300,000 cellsurface B7-H4 molecules (e.g., SK-BR-3 cells). In specific embodiments,an antibody or antigen-binding fragment thereof described herein, whichimmunospecifically binds to B7-H4 (e.g., human B7-H4) exhibitsantibody-dependent cellular cytotoxicity (ADCC) activity on cell lineswith at least 100,000 cell surface B7-H4 molecules (e.g., HCC 1569cells). In specific embodiments, an antibody or antigen-binding fragmentthereof described herein, which immunospecifically binds to B7-H4 (e.g.,human B7-H4) exhibits antibody-dependent cellular cytotoxicity (ADCC)activity on cell lines with at least 50,000 cell surface B7-H4 molecules(e.g., ZR-75-1 cells). In specific embodiments, an antibody orantigen-binding fragment thereof described herein, whichimmunospecifically binds to B7-H4 (e.g., human B7-H4) exhibitsantibody-dependent cellular cytotoxicity (ADCC) activity on cell lineswith at least 30,000 cell surface B7-H4 molecules (e.g., MDA-MB-468cells). In specific embodiments, an antibody or antigen-binding fragmentthereof described herein, which immunospecifically binds to B7-H4 (e.g.,human B7-H4) exhibits antibody-dependent cellular cytotoxicity (ADCC)activity on cell lines with at least 15,000 cell surface B7-H4 molecules(e.g., HCC1964 cells). Exemplary methods of measuring ADCC activity areprovided herein in Example 13.

In specific embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4), comprises framework regions (e.g., framework regions of the VHdomain and/or VL domain) that are human framework regions or derivedfrom human framework regions. Non-limiting examples of human frameworkregions are described in the art, e.g., see Kabat E A et al., (1991)supra). In certain embodiment, an antibody or antigen-binding fragmentthereof described herein comprises framework regions (e.g., frameworkregions of the VH domain and/or VL domain) that are primate (e.g.,non-human primate) framework regions or derived from primate (e.g.,non-human primate) framework regions.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which specifically binds to B7-H4 (e.g., human B7-H4),comprises one, two, or more VH framework regions (FRs) having the aminoacid sequences described herein for an antibody set forth in Table 5,supra (e.g., SEQ ID NOs: 465, 466, 467, and/or 469; SEQ ID NOs:235, 236,237 and/or 238; or SEQ ID NOs:265, 266, 267, and/or 268). In someembodiments, an antibody or antigen-binding fragment thereof describedherein, which specifically binds to B7-H4 (e.g., human B7-H4), comprisesone, two, or more VL framework regions (FRs) having the amino acidsequences described herein for an antibody set forth in Table 6, supra(e.g., SEQ ID NOs:239, 240, 241, and/or 242 or SEQ ID NOs:269, 270, 271,and/or 272). In specific embodiments, an antibody or antigen-bindingfragment thereof described herein, which specifically binds to B7-H4(e.g., human B7-H4), comprises one, two, or more VH framework regionshaving the amino acid sequences described herein for an antibody setforth in Table 5, supra, and one, two, or more VL framework regionshaving the amino acid sequences described herein for the same antibodyset forth in Table 6, supra (e.g., (i) SEQ ID NOs: 465, 466, 467, and/or469 and SEQ ID NOs:239, 240, 241, and/or 242; (ii) SEQ ID NOs:235, 236,237 and/or 238 and SEQ ID NOs:239, 240, 241, and/or 242 or (iii) SEQ IDNOs:265, 266, 267, and/or 268 and SEQ ID NOs:269, 270, 271, and/or 272).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which specifically binds to B7-H4 (e.g., human B7-H4),comprises VH framework regions (FRs) having at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the VH framework regions described herein in Table5, supra. In certain embodiments, an antibody or antigen-bindingfragment thereof described herein, which specifically binds to B7-H4(e.g., human B7-H4), comprises VL framework regions (FRs) having atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% sequence identity to the VL framework regionsdescribed herein Table 6, supra. In some embodiments, an antibody orantigen-binding fragment thereof described herein, which specificallybinds to B7-H4 (e.g., human B7-H4), comprises VH framework regions (FRs)having at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the VH frameworkregions described herein Table 5, supra, and VL framework regions (FRs)having at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the VL frameworkregions described herein Table 6, supra.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4), comprises a VH domain having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence of the VH domain of 20502or 22213 (e.g., SEQ ID NO:464 or 71), wherein the antibody comprises VHCDRs that are identical to the VH CDRs of 20502 or 22213 (e.g., SEQ IDNOs:458-460 or 65-67).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4), comprises a VL domain having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence of the VL domain of 20502or 22213 (e.g., SEQ ID NO:42 or 72), wherein the antibody orantigen-binding fragment thereof comprises VL CDRs that are identical tothe VL CDRs of 20502 or 22213 (e.g., SEQ ID NO:461-463 or 68-70).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4), comprises: (i) a VH domain having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence of the VH domain of 20502or 22213 (e.g., SEQ ID NO:464 or 71); and (ii) a VL domain having atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% sequence identity to the amino acid sequenceof the VL domain of 20502 or 22213 (e.g., SEQ ID NO:42 or 72), whereinthe antibody comprises VH CDRs and VL CDRs that are identical to the VHCDRs and VL CDRs of 20502 or 22213 (e.g., SEQ ID NOs:458-463 or SEQ IDNOs:65-70).

In another aspect, provided herein are antibodies or antigen-bindingfragments thereof that bind the same epitope of B7-H4 (e.g., an epitopeof human B7-H4) as an antibody or antigen-binding fragment thereofdescribed herein (e.g., 20502 or 22213).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4), comprises a VH domain having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence of the VH domain of 20502.1or 22213 (e.g., SEQ ID NO:41 or 71), wherein the antibody comprises VHCDRs that are identical to the VH CDRs of 20502.1 or 22213 (e.g., SEQ IDNOs: 35-37 or 65-67).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4), comprises a VL domain having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence of the VL domain of 20502.1or 22213 (e.g., SEQ ID NO:42 or 72), wherein the antibody orantigen-binding fragment thereof comprises VL CDRs that are identical tothe VL CDRs of 20502.1 or 22213 (e.g., SEQ ID NO:38-40 or 68-70).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to B7-H4 (e.g., humanB7-H4), comprises: (i) a VH domain having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence of the VH domain of 20502.1or 22213 (e.g., SEQ ID NO:41 or 71); and (ii) a VL domain having atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% sequence identity to the amino acid sequenceof the VL domain of 20502.1 or 22213 (e.g., SEQ ID NO:42 or 72), whereinthe antibody comprises VH CDRs and VL CDRs that are identical to the VHCDRs and VL CDRs of 20502.1 or 22213 (e.g., SEQ ID NOs:35-40 or SEQ IDNOs:65-70).

In another aspect, provided herein are antibodies or antigen-bindingfragments thereof that bind the same epitope of B7-H4 (e.g., an epitopeof human B7-H4) as an antibody or antigen-binding fragment thereofdescribed herein (e.g., 20502.1 or 22213).

Competition binding assays can be used to determine whether twoantibodies bind to overlapping epitopes. Competitive binding can bedetermined in an assay in which the immunoglobulin under test inhibitsspecific binding of a reference antibody to a common antigen, such asB7-H4. Numerous types of competitive binding assays are known, forexample: solid phase direct or indirect radioimmunoassay (RIA), solidphase direct or indirect enzyme immunoassay (EIA), sandwich competitionassay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253); solidphase direct biotin-avidin EIA (see Kirkland T N et al., (1986) JImmunol 137: 3614-9); solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see Harlow E & Lane D, (1988) Antibodies:A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using I-125 label (see Morel G A et al., (1988) Mol Immunol 25(1):7-15); solid phase direct biotin-avidin EIA (Cheung R C et al., (1990)Virology 176: 546-52); and direct labeled RIA. (Moldenhauer G et al.,(1990) Scand J Immunol 32: 77-82). Typically, such an assay involves theuse of purified antigen (e.g., B7-H4 such as human B7-H4) bound to asolid surface or cells bearing either of these, an unlabeled testimmunoglobulin and a labeled reference immunoglobulin. Competitiveinhibition can be measured by determining the amount of label bound tothe solid surface or cells in the presence of the test immunoglobulin.Usually the test immunoglobulin is present in excess. Usually, when acompeting antibody is present in excess, it will inhibit specificbinding of a reference antibody to a common antigen by at least 50-55%,55-60%, 60-65%, 65-70%, 70-75% or more. A competition binding assay canbe configured in a large number of different formats using eitherlabeled antigen or labeled antibody. In a common version of this assay,the antigen is immobilized on a 96-well plate. The ability of unlabeledantibodies to block the binding of labeled antibodies to the antigen isthen measured using radioactive or enzyme labels. For further detailssee, for example, Wagener C et al., (1983) J Immunol 130: 2308-2315;Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M et al.,(1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407 andAntibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, pp.386-389.

In one embodiment, a competition assay is performed using surfaceplasmon resonance (BTAcore®), e.g., by an ‘in tandem approach’ such asthat described by Abdiche Y N et al., (2009) Analytical Biochem 386:172-180, whereby B7-H4 antigen is immobilized on the chip surface, forexample, a CMS sensor chip and the anti-B7-H4 antibodies are then runover the chip. To determine if an antibody or antigen-binding fragmentthereof competes with an anti-B7-H4 antibody described herein, theanti-B7-H4 antibody is first run over the chip surface to achievesaturation and then the potential, competing antibody is added. Bindingof the competing antibody or antigen-binding fragment thereof can thenbe determined and quantified relative to a non-competing control.

In one embodiment, Fortebio Octet competition binding (e.g., asdescribed in Example 2 below) is used to determine that a B7-H4 antibodyor antigen-binding fragment thereof competitively inhibits the bindingof another B7-H4 antibody or antigen-binding fragment thereof to B7-H4.

In another aspect, provided herein are antibodies that competitivelyinhibit (e.g., in a dose dependent manner) an antibody orantigen-binding fragment thereof described herein (e.g., 20502, 20502.1or 22213) from binding to B7-H4 (e.g., human B7-H4), as determined usingassays known to one of skill in the art or described herein (e.g., ELISAcompetitive assays, or suspension array or surface plasmon resonanceassay).

In specific aspects, provided herein is an antibody or antigen-bindingfragment which competitively inhibits (e.g., in a dose dependent manner)binding to B7-H4 (e.g., human B7-H4), of an antibody comprising a VHdomain having the amino acid sequence set forth in SEQ ID NO:464, and aVL domain having the amino acid sequence set for the in SEQ ID NO:42.

In specific aspects, provided herein is an antibody or antigen-bindingfragment which competitively inhibits (e.g., in a dose dependent manner)binding to B7-H4 (e.g., human B7-H4), of an antibody comprising a VHdomain having the amino acid sequence set forth in SEQ ID NO:41, and aVL domain having the amino acid sequence set for the in SEQ ID NO:42.

In specific aspects, provided herein is an antibody or antigen-bindingfragment which competitively inhibits (e.g., in a dose dependent manner)binding to B7-H4 (e.g., human B7-H4), of an antibody comprising a VHdomain having the amino acid sequence set forth in SEQ ID NO:71, and aVL domain having the amino acid sequence set for the in SEQ ID NO:72.

In specific aspects, provided herein is an antibody or antigen-bindingfragment thereof which competitively inhibits (e.g., in a dose dependentmanner) binding to B7-H4 (e.g., human B7-H4), with an antibodycomprising (i) a VH domain comprising a VH CDR1, VH CDR2, and VH CDR3having the amino acid sequences of the VH CDRs listed in Table 1; and(ii) a VL domain comprising a VL CDR1, VL CDR2, and VL CDR3 having theamino acid sequences of the CDRs listed in Table 2.

In specific aspects, provided herein is an antibody or antigen-bindingfragment thereof, which immunospecifically binds to the same B7-H4(e.g., human B7-H4) epitope as that of 20502, 20502.1, or 22213.

In another specific embodiment, an antibody or antigen-binding fragmentthereof described herein, immunospecifically binds to the same B7-H4(e.g., human B7-H4) epitope as that of an antibody comprising (i) a VHdomain comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acidsequences of the CDRs listed in Table 1 and (ii) a VL domain comprisinga VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of theCDRs listed in Table 2.

In a specific aspect, an antigen-binding fragment as described herein,which immunospecifically binds to B7-H4 (e.g., human B7-H4), is selectedfrom the group consisting of a Fab, Fab′, F(ab′)2, and scFv, wherein theFab, Fab′, F(ab′)2, or scFv comprises a heavy chain variable regionsequence and a light chain variable region sequence of an anti-B7-H4antibody or antigen-binding fragment thereof as described herein. A Fab,Fab′, F(ab′)2, or scFv can be produced by any technique known to thoseof skill in the art, including, but not limited to, those discussed inSection 5.3, infra. In certain embodiments, the Fab, Fab′, F(ab′)2, orscFv further comprises a moiety that extends the half-life of theantibody in vivo. The moiety is also termed a “half-life extendingmoiety.” Any moiety known to those of skill in the art for extending thehalf-life of a Fab, Fab′, F(ab′)2, or scFv in vivo can be used. Forexample, the half-life extending moiety can include a Fc region, apolymer, an albumin, or an albumin binding protein or compound. Thepolymer can include a natural or synthetic, optionally substitutedstraight or branched chain polyalkylene, polyalkenylene,polyoxylalkylene, polysaccharide, polyethylene glycol, polypropyleneglycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose,dextran, glycogen, or derivative thereof. Substituents can include oneor more hydroxy, methyl, or methoxy groups. In certain embodiments, theFab, Fab′, F(ab′)2, or scFv can be modified by the addition of one ormore C-terminal amino acids for attachment of the half-life extendingmoiety. In certain embodiments the half-life extending moiety ispolyethylene glycol or human serum albumin. In certain embodiments, theFab, Fab′, F(ab′)2, or scFv is fused to a Fc region.

An anti-B7-H4 antibody or antigen-binding fragment thereof can be fusedor conjugated (e.g., covalently or noncovalently linked) to a detectablelabel or substance. Examples of detectable labels or substances includeenzyme labels, such as, glucose oxidase; radioisotopes, such as iodine(125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In),and technetium (99Tc); luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin. Suchlabeled antibodies or antigen-binding fragments thereof can be used todetect B7-H4 (e.g., human B7-H4) protein. See, e.g., Section 5.5.2,infra.

Antibody Production

Antibodies and antigen-binding fragments thereof that immunospecificallybind to B7-H4 (e.g., human B7-H4) can be produced by any method known inthe art for the synthesis of antibodies and antigen-binding fragmentsthereof, for example, by chemical synthesis or by recombinant expressiontechniques. The methods described herein employ, unless otherwiseindicated, conventional techniques in molecular biology, microbiology,genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR,oligonucleotide synthesis and modification, nucleic acid hybridization,and related fields within the skill of the art. These techniques aredescribed, for example, in the references cited herein and are fullyexplained in the literature. See, e.g., Sambrook J et al., (2001)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Ausubel F M et al., Current Protocolsin Molecular Biology, John Wiley & Sons (1987 and annual updates);Current Protocols in Immunology, John Wiley & Sons (1987 and annualupdates) Gait (ed.) (1984) Oligonucleotide Synthesis: A PracticalApproach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides andAnalogues: A Practical Approach, IRL Press; Birren B et al., (eds.)(1999) Genome Analysis: A Laboratory Manual, Cold Spring HarborLaboratory Press.

In a certain aspect, provided herein is a method of making an antibodyor antigen-binding fragment which immunospecifically binds to B7-H4(e.g., human B7-H4) comprising culturing a cell or host cell describedherein. In a certain aspect, provided herein is a method of making anantibody or antigen-binding fragment thereof which immunospecificallybinds to B7-H4 (e.g., human B7-H4) comprising expressing (e.g.,recombinantly expressing) the antibody or antigen-binding fragmentthereof using a cell or host cell described herein (e.g., a cell or ahost cell comprising polynucleotides encoding an antibody orantigen-binding fragment thereof described herein). In a particularembodiment, the cell is an isolated cell. In a particular embodiment,the exogenous polynucleotides have been introduced into the cell. In aparticular embodiment, the method further comprises the step ofpurifying the antibody or antigen-binding fragment obtained from thecell or host cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York).

Monoclonal antibodies or antigen-binding fragments thereof can beprepared using a wide variety of techniques known in the art includingthe use of hybridoma, recombinant, and phage display technologies,yeast-based presentation technologies, or a combination thereof. Forexample, monoclonal antibodies or antigen-binding fragments thereof canbe produced using hybridoma techniques including those known in the artand taught, for example, in Harlow E & Lane D, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); HammerlingG J et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681(Elsevier, N.Y., 1981), or as described in Kohler G & Milstein C (1975)Nature 256: 495. Examples of yeast-based presentation methods that canbe employed to select and generate the antibodies described hereininclude those disclosed in, for example, WO2009/036379A2; WO2010/105256;and WO2012/009568, each of which is herein incorporated by reference inits entirety.

In specific embodiments, a monoclonal antibody or antigen-bindingfragment is an antibody or antigen-binding fragment produced by a clonalcell (e.g., hybridoma or host cell producing a recombinant antibody orantigen-binding fragment), wherein the antibody or antigen-bindingfragment immunospecifically binds to B7-H4 (e.g., human B7-H4) asdetermined, e.g., by ELISA or other antigen-binding assays known in theart or in the Examples provided herein. In particular embodiments, amonoclonal antibody or antigen-binding fragment thereof can be achimeric or a humanized antibody or antigen-binding fragment thereof. Incertain embodiments, a monoclonal antibody or antigen-binding fragmentthereof can be a Fab fragment or a F(ab′)2 fragment. Monoclonalantibodies or antigen-binding fragments thereof described herein can,for example, be made by the hybridoma method as described in Kohler G &Milstein C (1975) Nature 256: 495 or can, e.g., be isolated from phagelibraries using the techniques as described herein, for example. Othermethods for the preparation of clonal cell lines and of monoclonalantibodies and antigen-binding fragments thereof expressed thereby arewell known in the art (see, for example, Chapter 11 in: Short Protocolsin Molecular Biology, (2002) 5th Ed., Ausubel F M et al., supra).

Antigen-binding fragments of antibodies described herein can begenerated by any technique known to those of skill in the art. Forexample, Fab and F(ab′)2 fragments described herein can be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments). A Fab fragment corresponds to one of the two identical armsof a tetrameric antibody molecule and contains the complete light chainpaired with the VH and CH1 domains of the heavy chain. A F(ab′)2fragment contains the two antigen-binding arms of a tetrameric antibodymolecule linked by disulfide bonds in the hinge region.

Further, the antibodies or antigen-binding fragments thereof describedherein can also be generated using various phage display and/oryeast-based presentation methods known in the art. In phage displaymethods, proteins are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In particular, DNAsequences encoding VH and VL domains are amplified from animal cDNAlibraries (e.g., human or murine cDNA libraries of affected tissues).The DNA encoding the VH and VL domains are recombined together with ascFv linker by PCR and cloned into a phagemid vector. The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13, and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antibody orantigen-binding fragment thereof that binds to a particular antigen canbe selected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Examples of phagedisplay methods that can be used to make the antibodies or fragmentsdescribed herein include those disclosed in Brinkman U et al., (1995) JImmunol Methods 182: 41-50; Ames R S et al., (1995) J Immunol Methods184: 177-186; Kettleborough C A et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187: 9-18; Burton D R & Barbas C F(1994) Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134;International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO 97/13844; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727, 5,733,743, and 5,969,108.

A humanized antibody or antigen-binding fragment thereof can be selectedfrom any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE,and any isotype, including IgG1, IgG2, IgG3 and IgG4.

1.2.1 Polynucleotides

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody or antigen-binding fragmentthereof described herein or a domain thereof (e.g., a variable lightchain region and/or variable heavy chain region) that immunospecificallybinds to a B7-H4 (e.g., human B7-H4) antigen, and vectors, e.g., vectorscomprising such polynucleotides for recombinant expression in host cells(e.g., E. coli and mammalian cells).

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies or antigen-binding fragmentsthereof, which immunospecifically bind to a B7-H4 polypeptide (e.g.,human B7-H4) and comprise an amino acid sequence as described herein, aswell as antibodies or antigen-binding fragments that compete with suchantibodies or antigen-binding fragments for binding to a B7-H4polypeptide (e.g., in a dose-dependent manner), or which bind to thesame epitope as that of such antibodies or antigen-binding fragments.

Also provided herein is a polynucleotide comprising a nucleotidesequence encoding a polypeptide comprising a sequence selected from thegroup consisting of SEQ ID NOs:11, 12, 21, 22, 31, 32, 41, 464, 42, 51,52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131,132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201,and 202. In some embodiments, an antibody or antigen-binding fragmentthereof comprising the polypeptide immunospecifically binds to B7-H4.

Also provided herein is a polynucleotide comprising a nucleotidesequence encoding a polypeptide comprising a sequence selected from thegroup consisting of SEQ ID NOs:13, 14, 23, 24, 33, 34, 43, 469, 44, 53,54, 63, 64, 73, 74, 83, 84, 93, 94, 103, 104, 113, 114, 123, 124, 133,134, 143, 144, 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203,and 204. In some embodiments, an antibody or antigen-binding fragmentthereof comprising the polypeptide immunospecifically binds to B7-H4.

Also provided herein are polynucleotides comprising a variable heavychain-encoding nucleotide sequence shown in Table 9, e.g., wherein anantibody or antigen-binding fragment thereof comprising the encodedvariable heavy chain binds to B7-H4.

TABLE 9 Variable heavy chain-encoding polynucleotide sequencesVariable Heavy Chain-Encoding AntibodyPolynucleotide Sequence (SEQ ID NO) 15461CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAACATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAAGGATCTTACCCCAATTGGTTTGATCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 213) 20500CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAAGAGTGGTAGTCACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAACATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAGGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGGGAAGGATCTTACCCCAATTGGTTTGATCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 223) 20501CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAAGAGTGGTAGTCACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAACATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAAGGATCTTACCCCAATTGGTTGGATCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 233) 20502CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAAAAGTGGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAACATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAGAAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAAGGATCTTACCCCAATCAGTTTGATCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 470) 20502.1CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAAGAGTGGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAACATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAAGGATCTTACCCCAATCAGTTTGATCCATGGGGACAGGGTATATTGGTCACCGTCTCCTCA (SEQ ID NO: 243) 22208CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAAGAGTGGTAGTCACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAACATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATGTCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAAGGATCTTACCCCAATTGGTTTGATCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 253) 15462CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAACATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAAGGATCTTACACAACCGTGTTAAACGTATGGGGTCAGGGTACAATGGTCACCGTCTCCTCA (SEQ ID NO: 263) 22213CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCGGGAGGGGGAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAACATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAAGGATCTTACACAACCGTGTTAAACGTATGGGGTCAGGGTACAATGGTCACCGTCTCCTCA (SEQ ID NO: 273) 15465CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGTACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGAACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAATCTAGCACCATATCTGCCGACTTCGACCTATGGGGGAGAGGTACCTTGGTCACCGTCTCCTCA (SEQ ID NO: 283) 20506CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGTACTGCCTCTGGTGGCTCCATCAGCCATGGTGGGTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGAACATCTATTACAGTGGGAGCACCTACTACAATCCGTCCCTCAAGAGTCGAGTTACCATGTCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAATCTAGCACCATATCTGCCGACTTCGACCTATGGGGGAGAGGTACCTTGGTCACCGTCTCCTCA (SEQ ID NO: 293) 15483CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGTACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGAACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGGGTTGAGCACCATAGACGAGGCATTCGACCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 303) 20513CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCGATGGTAGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGAACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAGGAGTCGAGTTACCATGTCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGGGTTGAGCACCATAGACGAGGCATTCGACCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 313) 22216CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGTACTGTCTCTGGTGGCTCCATCAGCGATGGTAGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGAACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAGGAGTCGAGTTACCATGTCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGGGTTGAGCACCATAGACGAGGCATTCGACCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 323) 15489CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATAGTAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGGCTCTGGACAGTATGCAGCTCCTGATTATGGAATGGACGTATGGGGCCAGGGAACAACTGTCACCGTCTCCTCA (SEQ ID NO: 333) 20516CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCATTAGTTACTACTGGGGGTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTCTAGTGGGAGCACCTCGTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGGCTCTGGACTGTATGCAGCTCCTGATTATGGACTTGACGTATGGGGTCAGGGAACAACTGTCACCGTCTCCTCA (SEQ ID NO: 343) 15472GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACCATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAGAGGTGCCGGACACTACGACCTCGTCGGACGATACTGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 353) 15503GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAGAGTGGGATTCAGAGCATTAAACTACTGGGGACAGGGTACAACTGTCACCGTCTCCTCA (SEQ ID NO: 363) 15495CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTGCGCAAGACAGCAATACGACGGTAGACGATACTTCGGCCTATGGGGGAGAGGTACCTTGGTCACCGTCTCCTCA (SEQ ID NO: 373) 15478CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTGCGCCAGAGGTGGGCCTTGGTTTGATCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 383) 15441GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATCCTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGCCTTCTTTGGCAACAATGTTAGCCTTCGATATCTGGGGTCAGGGTACAATGGTCACCGTCTCCTCA (SEQ ID NO: 393) 20496CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTGTTTACTACTGGAGTTGGATCCGCCAGCCCCCAGGGAAGGGGTTGGAGTGGATTGGGAGTATCCTGGTGAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGCTGTATCCTTCTTAGACGTATGGGGTCAGGGTACAATGGTCATCGTCTCCTCA (SEQ ID NO: 403)

Also provided herein are polynucleotides comprising a variable lightchain-encoding nucleotide sequence shown in Table 10, e.g., wherein anantibody or antigen-binding fragment thereof comprising the encodedvariable light chain binds to B7-H4.

TABLE 10 Variable light chain-encoding polynucleotide sequencesVariable Light Chain-Encoding AntibodyPolynucleotide Sequence (SEQ ID NO) 15461GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTACCACTCCTTCCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 214) 20500GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTACCACTCCTTCCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 224) 20501GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTACCACTCCTTCCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 234) 20502 andGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGG 20502.1GAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTACCACTCCTTCCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 244) 22208GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGTCCGTTAGCACCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGACGCATCCGCCAGGGTCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTACCACTCCTTCCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 254) 15462GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGCCAGTTACCCTCTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 264) 22213GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGCCAGCAGCCACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGACGCAGTCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGCCAGTTACCCTCTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 274) 15465GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGGCACACACCTTCCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 284) 20506GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGGCACACACCTTCCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 294) 15483GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAAGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGGACAACAGTTACCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 304) 20513GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAAGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGGACAACAGTTACCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 314) 22216GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTAAAAGTATTAGTTCCTGGTTGGCCTGGTATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGAAGCCTCCTCCTTGCACAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGGACAACAGTTACCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 324) 15489GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAAGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGGACAATAGCTTCCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 334) 20516GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAAGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGGACAATAGCTTCCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 344) 15472GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAACTATACAGTCTCCCTCCTACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 354) 15503GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGGCAACCAGTTACCCTCCTTGGACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 364) 15495GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAGCGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGGTCAACGTCTGGCCTCCTACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 374) 15478GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAAGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGTACAATAGCTACCCTCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 384) 15441GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGTCCAAAAGTTACCCTAGGACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 394) 20496GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAAAGCTACGACCCCCCTTGGACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 404)

Also provided herein are polynucleotides comprising the nucleotidesequence of SEQ ID NO:213, 214, 223, 224, 233, 234, 243, 470, 244, 253,254, 263, 264, 273, 274, 283, 284, 293, 294, 303, 304, 313, 314, 323,324, 333, 334, 343, 344, 353, 354, 363, 364, 373, 374, 383, 384, 393,394, 403, or 404. Also provided herein are polynucleotides comprising(i) the nucleotide sequence of SEQ ID NO:213, 214, 223, 224, 233, 234,243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293, 294, 303,304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363, 364, 373,374, 383, 384, 393, 394, 403, or 404 and (ii) the nucleotide sequence ofSEQ ID NO:408 or 406.

Also provided herein are polynucleotides comprising a nucleotidesequence that encodes SEQ ID NO:11, 12, 21, 22, 31, 32, 41, 464, 42, 51,52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131,132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201, or202 and is at least about 80%, 85%, or 90% identical to SEQ ID NO:213,214, 223, 224, 233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274,283, 284, 293, 294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344,353, 354, 363, 364, 373, 374, 383, 384, 393, 394, 403, or 404,respectively.

Also provided herein are polynucleotides comprising a nucleotidesequence that encodes SEQ ID NO:11, 12, 21, 22, 31, 32, 41, 464, 42, 51,52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131,132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201, or202 and is at least about 95% identical to SEQ ID NO:213, 214, 223, 224,233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293,294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363,364, 373, 374, 383, 384, 393, 394, 403, or 404, respectively.

Also provided herein are polynucleotides comprising a nucleotidesequence that encodes SEQ ID NO:11, 12, 21, 22, 31, 32, 41, 464, 42, 51,52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131,132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201, or202 and is at least about 96% identical to SEQ ID NO:213, 214, 223, 224,233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293,294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363,364, 373, 374, 383, 384, 393, 394, 403, or 404, respectively.

Also provided herein are polynucleotides comprising a nucleotidesequence that encodes SEQ ID NO:11, 12, 21, 22, 31, 32, 41, 464, 42, 51,52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131,132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201, or202 and is at least about 97% identical to SEQ ID NO:213, 214, 223, 224,233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293,294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363,364, 373, 374, 383, 384, 393, 394, 403, or 404, respectively.

Also provided herein are polynucleotides comprising a nucleotidesequence that encodes SEQ ID NO:11, 12, 21, 22, 31, 32, 41, 464, 42, 51,52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131,132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201, or202 and is at least about 98% identical to SEQ ID NO:213, 214, 223, 224,233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293,294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363,364, 373, 374, 383, 384, 393, 394, 403, or 404, respectively.

Also provided herein are polynucleotides comprising a nucleotidesequence that encodes SEQ ID NO:11, 12, 21, 22, 31, 32, 41, 464, 42, 51,52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131,132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201, or202 and is at least about 99% identical to SEQ ID NO:213, 214, 223, 224,233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293,294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363,364, 373, 374, 383, 384, 393, 394, 403, or 404, respectively.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody or an antigen-binding fragment described herein. Thepolynucleotides can comprise nucleotide sequences encoding a heavy chaincomprising the VH FRs and CDRs of antibodies described herein (see,e.g., Tables 1 and 5). The polynucleotides can comprise nucleotidesequences encoding a light chain comprising the VL FRs and CDRs ofantibodies described herein (see, e.g., Tables 2 and 6).

In specific embodiments, a polynucleotide described herein encodes a VHdomain comprising the amino acid sequence set forth in SEQ ID NO:464. Inspecific embodiments, a polynucleotide described herein encodes a VLdomain comprising the amino acid sequence set forth in SEQ ID NO:42.

In specific embodiments, a polynucleotide described herein encodes a VHdomain comprising the amino acid sequence set forth in SEQ ID NO:41. Inspecific embodiments, a polynucleotide described herein encodes a VLdomain comprising the amino acid sequence set forth in SEQ ID NO:42.

In specific embodiments, a polynucleotide described herein encodes a VHdomain comprising the amino acid sequence set forth in SEQ ID NO:71. Inspecific embodiments, a polynucleotide described herein encodes a VLdomain comprising the amino acid sequence set forth in SEQ ID NO:72.

In particular embodiments, provided herein are polynucleotidescomprising a nucleotide sequence encoding an anti-B7-H4 antibodycomprising three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, VH VLCDR3 of any one of antibodies described herein (e.g., see Table 1). Inspecific embodiments, provided herein are polynucleotides comprisingthree VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 ofany one of antibodies described herein (e.g., see Table 2). In specificembodiments, provided herein are polynucleotides comprising a nucleotidesequence encoding an anti-B7-H4 antibody comprising three VH chain CDRs,e.g., containing VH CDR1, VH CDR2, and VH CDR3 of any one of antibodiesdescribed herein (e.g., see Table 1) and three VL chain CDRs, e.g.,containing VL CDR1, VL CDR2, and VL CDR3 of any one of antibodiesdescribed herein (e.g., see Table 2).

In particular embodiments, provided herein are polynucleotidescomprising a nucleotide sequence encoding an anti-B7-H4 antibody or anantigen-binding fragment thereof or a fragment thereof comprising a VHdomain, e.g., containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, comprising anamino acid sequence described herein (e.g., see Tables 1 and 5, e.g.,the VH CDRs and VH FRs of a particular antibody identified by name inthe tables). In specific embodiments, provided herein arepolynucleotides comprising a nucleotide sequence encoding an anti-B7-H4antibody or antigen-binding fragment thereof or a fragment thereofcomprising a VL domain, e.g., containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4,comprising an amino acid sequence described herein (e.g., see Tables 2and 6, e.g., the VL CDRs and VL FRs of a particular antibody identifiedby name in the Tables).

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding a heavy chain variable region comprising anamino acid sequence described herein (e.g., SEQ ID NO:464, 41, or 71),wherein an antibody containing the heavy chain variable regionimmunospecifically binds to B7-H4 (e.g., human B7-H4). In a certainembodiment, a polynucleotide described herein comprises a heavy chainvariable region-encoding sequence provided herein (e.g., the variableregion-encoding portion of SEQ ID NO:470, 243, or 273), wherein anantibody containing the heavy chain variable region immunospecificallybinds to B7-H4 (e.g., human B7-H4).

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding a light chain variable region comprising anamino acid sequence described herein (e.g., SEQ ID NO:42 or 72), whereinan antibody containing the light chain variable regionimmunospecifically binds to B7-H4 (e.g., human B7-H4). In a certainembodiment, a polynucleotide described herein comprises a light chainvariable region-encoding sequence provided herein (e.g., the variableregion-encoding portion of SEQ ID NO:244 or 274), wherein an antibodycontaining the heavy chain variable region immunospecifically binds toB7-H4 (e.g., human B7-H4).

In a particular embodiment, a polynucleotide or combination ofpolynucleotides provided herein comprises a nucleotide sequence orcombination of nucleotide sequences encoding an antibody orantigen-binding fragment thereof that immunospecifically binds to B7-H4(e.g., human B7-H4), wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain, wherein the heavy chain comprises aheavy chain variable domain comprising an amino acid sequence set forthin Table 3 (e.g., SEQ ID NO:464, 41, or 71) and a constant regioncomprising the amino acid sequence of a human gamma (γ) heavy chainconstant region.

In a particular embodiment, a polynucleotide or combination ofpolynucleotides provided herein comprises a nucleotide sequence orcombination of nucleotide sequences encoding an antibody orantigen-binding fragment thereof that immunospecifically binds to B7-H4(e.g., human B7-H4), wherein the antibody or antigen-binding fragmentthereof comprises a light chain, wherein the light chain comprises alight chain variable domain comprising an amino acid sequence set forthin Table 4 (e.g., SEQ ID NO:42 or 72) and a constant region comprisingthe amino acid sequence of a human lambda light chain constant region.

In a particular embodiment, a polynucleotide or combination ofpolynucleotides provided herein comprises a nucleotide sequence orcombination of nucleotide sequences encoding an antibody orantigen-binding fragment thereof that immunospecifically binds to B7-H4(e.g., human B7-H4), wherein the antibody or antigen-binding fragmentthereof comprises (i) a heavy chain, wherein heavy chain comprises aheavy chain variable domain comprising an amino acid sequence set forthin Table 3 (e.g., SEQ ID NO:464, 41, or 71) and a constant regioncomprising the amino acid sequence of a human gamma (γ) heavy chainconstant region and (ii) a light chain, wherein light chain comprises alight chain variable domain comprising an amino acid sequence set forthin Table 4 (e.g., SEQ ID NO:42 or 72) and a constant region comprisingthe amino acid sequence of a human lambda light chain constant region.

In one embodiment, a combination of polynucleotides provided hereincomprises a polynucleotide comprising the nucleotide sequence of SEQ IDNO:470 and a polynucleotide comprising the nucleotide sequence of SEQ IDNO:244.

In one embodiment, a combination of polynucleotides provided hereincomprises a polynucleotide comprising the nucleotide sequence of SEQ IDNO:243 and a polynucleotide comprising the nucleotide sequence of SEQ IDNO:244.

In one embodiment, a combination of polynucleotides provided hereincomprises a polynucleotide comprising the nucleotide sequence of SEQ IDNO:273 and a polynucleotide comprising the nucleotide sequence of SEQ IDNO:274.

In a specific embodiment, provided herein are polynucleotides comprisinga nucleotide sequence encoding an anti-B7-H4 antibody or antigen-bindingfragment thereof or a domain thereof, designated herein, see, e.g.,Tables 1-8.

Also provided herein are polynucleotides encoding an anti-B7-H4 antibodyor antigen-binding fragment thereof described herein or a domain thereofthat are optimized, e.g., by codon/RNA optimization, replacement withheterologous signal sequences, and elimination of mRNA instabilityelements. Methods to generate optimized nucleic acids encoding ananti-B7-H4 antibody or antigen-binding fragment thereof or a domainthereof (e.g., heavy chain, light chain, VH domain, or VL domain) forrecombinant expression by introducing codon changes (e.g., a codonchange that encodes the same amino acid due to the degeneracy of thegenetic code) and/or eliminating inhibitory regions in the mRNA can becarried out by adapting the optimization methods described in, e.g.,U.S. Pat. Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and6,794,498, accordingly.

A polynucleotide encoding an antibody or antigen-binding fragmentthereof described herein or a domain thereof can be generated fromnucleic acid from a suitable source (e.g., a hybridoma) using methodswell known in the art (e.g., PCR and other molecular cloning methods).For example, PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of a known sequence can be performed using genomicDNA obtained from hybridoma cells producing the antibody of interest.Such PCR amplification methods can be used to obtain nucleic acidscomprising the sequence encoding the light chain and/or heavy chain ofan antibody or antigen-binding fragment thereof. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody or antigen-binding fragment thereof. The amplifiednucleic acids can be cloned into vectors for expression in host cellsand for further cloning, for example, to generate chimeric and humanizedantibodies or antigen-binding fragments thereof.

Polynucleotides provided herein can be, e.g., in the form of RNA or inthe form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, andDNA can be double-stranded or single-stranded. If single stranded, DNAcan be the coding strand or non-coding (anti-sense) strand. In certainembodiments, the polynucleotide is a cDNA or a DNA lacking one moreendogenous introns. In certain embodiments, a polynucleotide is anon-naturally occurring polynucleotide. In certain embodiments, apolynucleotide is recombinantly produced. In certain embodiments, thepolynucleotides are isolated. In certain embodiments, thepolynucleotides are substantially pure. In certain embodiments, apolynucleotide is purified from natural components.

1.2.2 Cells and Vectors

In certain aspects, provided herein are vectors (e.g., expressionvectors) comprising polynucleotides comprising nucleotide sequencesencoding anti-B7-H4 antibodies and antigen-binding fragments thereof ora domain thereof for recombinant expression in host cells, preferably inmammalian cells. Also provided herein are cells, e.g. host cells,comprising such vectors for recombinantly expressing anti-B7-H4antibodies or antigen-binding fragments thereof described herein (e.g.,human or humanized antibodies or antigen-binding fragments thereof). Ina particular aspect, provided herein are methods for producing anantibody or antigen-binding fragments thereof described herein,comprising expressing such antibody or antigen-binding fragment thereofin a host cell.

In certain embodiments, recombinant expression of an antibody orantigen-binding fragment thereof or domain thereof described herein(e.g., a heavy or light chain described herein) that specifically bindsto B7-H4 (e.g., human B7-H4) involves construction of an expressionvector containing a polynucleotide that encodes the antibody orantigen-binding fragment thereof or domain thereof. Once apolynucleotide encoding an antibody or antigen-binding fragment thereofor domain thereof (e.g., heavy or light chain variable domain) describedherein has been obtained, the vector for the production of the antibodyor antigen-binding fragment thereof can be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody or antigen-binding fragment thereof or domain thereof (e.g.,light chain or heavy chain) encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody orantigen-binding fragment thereof or domain thereof (e.g., light chain orheavy chain) coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Also provided are replicable vectors comprising anucleotide sequence encoding an antibody or antigen-binding fragmentthereof described herein, a heavy or light chain, a heavy or light chainvariable domain, or a heavy or light chain CDR, operably linked to apromoter. Such vectors can, for example, include the nucleotide sequenceencoding the constant region of the antibody or antigen-binding fragmentthereof (see, e.g., International Publication Nos. WO 86/05807 and WO89/01036; and U.S. Pat. No. 5,122,464), and variable domains of theantibody or antigen-binding fragment thereof can be cloned into such avector for expression of the entire heavy, the entire light chain, orboth the entire heavy and light chains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody or antigen-bindingfragment thereof described herein (e.g., an antibody or antigen-bindingfragment thereof comprising the six CDRs, the VH, the VL, the VH and theVL, the heavy chain, the light chain, or the heavy and the light chainof 20502, 20502.1, or 22213) or a domain thereof (e.g., the VH, the VL,the VH and the VL, the heavy chain, or the light chain of 20502,20502.1, or 22213). Thus, provided herein are host cells containing apolynucleotide encoding an antibody or antigen-binding fragment thereofdescribed herein (e.g., an antibody or antigen-binding fragment thereofcomprising the six CDRs, the VH, the VL, the VH and the VL, the heavychain, the light chain, or the heavy and the light chain of 20502,20502.1, or 22213) or a domain thereof (e.g., the VH, the VL, the VH andthe VL, the heavy chain, or the light chain of 20502, 20502.1, or22213), operably linked to a promoter for expression of such sequencesin the host cell. In certain embodiments, for the expression ofdouble-chained antibodies or antigen-binding fragments thereof, vectorsencoding both the heavy and light chains, individually, can beco-expressed in the host cell for expression of the entireimmunoglobulin, as detailed below. In certain embodiments, a host cellcontains a vector comprising a polynucleotide encoding both the heavychain and light chain of an antibody described herein (e.g., the heavyand the light chain of 20502, 20502.1, or 22213), or a domain thereof(e.g., the VH and the VL of 20502, 20502.1, or 22213). In specificembodiments, a host cell contains two different vectors, a first vectorcomprising a polynucleotide encoding a heavy chain or a heavy chainvariable region of an antibody or antigen-binding fragment thereofdescribed herein, and a second vector comprising a polynucleotideencoding a light chain or a light chain variable region of an antibodydescribed herein (e.g., an antibody comprising the six CDRs of 20502,20502.1, or 22213), or a domain thereof. In other embodiments, a firsthost cell comprises a first vector comprising a polynucleotide encodinga heavy chain or a heavy chain variable region of an antibody orantigen-binding fragment thereof described herein, and a second hostcell comprises a second vector comprising a polynucleotide encoding alight chain or a light chain variable region of an antibody orantigen-binding fragment thereof described herein (e.g., an antibody orantigen-binding fragment thereof comprising the six CDRs of 20502,20502.1, or 22213). In specific embodiments, a heavy chain/heavy chainvariable region expressed by a first cell associated with a lightchain/light chain variable region of a second cell to form an anti-B7-H4antibody or antigen-binding fragment thereof described herein (e.g.,antibody or antigen-binding fragment thereof comprising the six CDRs of20502, 20502.1, or 22213). In certain embodiments, provided herein is apopulation of host cells comprising such first host cell and such secondhost cell.

In a particular embodiment, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-B7-H4 antibody orantigen-binding fragment thereof described herein, and a second vectorcomprising a polynucleotide encoding a heavy chain/heavy chain variableregion of an anti-B7-H4 antibody or antigen-binding fragment thereofdescribed herein (e.g., antibody or antigen-binding fragment thereofcomprising the CDRs of 20502, 20502.1, or 22213). Alternatively, asingle vector can be used which encodes, and is capable of expressing,both heavy and light chain polypeptides.

A variety of host-expression vector systems can be utilized to expressantibodies and antigen-binding fragments thereof described herein (e.g.,an antibody or antigen-binding fragment thereof comprising the CDRs of20502, 20502.1, or 22213) (see, e.g., U.S. Pat. No. 5,807,715). Suchhost-expression systems represent vehicles by which the coding sequencesof interest can be produced and subsequently purified, but alsorepresent cells which can, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody orantigen-binding fragment thereof described herein in situ. These includebut are not limited to microorganisms such as bacteria (e.g., E. coliand B. subtilis) transformed with recombinant bacteriophage DNA, plasmidDNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems (e.g., green algae such as Chlamydomonasreinhardtii) infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing antibody coding sequences; or mammalian cell systems(e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6,VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210,R1.1, B-W, L-M, BSC1, BSC40, YB/20 and BMT10 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). In a specific embodiment, cells for expressingantibodies and antigen-binding fragments thereof described herein (e.g.,an antibody or antigen-binding fragment thereof comprising the CDRs of20502, 20502.1, or 22213) are CHO cells, for example CHO cells from theCHO GS System™ (Lonza). In a particular embodiment, cells for expressingantibodies described herein are human cells, e.g., human cell lines. Ina specific embodiment, a mammalian expression vector is pOptiVEC™ orpcDNA3.3. In a particular embodiment, bacterial cells such asEscherichia coli, or eukaryotic cells (e.g., mammalian cells),especially for the expression of whole recombinant antibody molecule,are used for the expression of a recombinant antibody molecule. Forexample, mammalian cells such as Chinese hamster ovary (CHO) cells inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies (Foecking M K & Hofstetter H (1986) Gene 45:101-105; and Cockett M I et al., (1990) Biotechnology 8: 662-667). Incertain embodiments, antibodies or antigen-binding fragments thereofdescribed herein are produced by CHO cells or NS0 cells.

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products cancontribute to the function of the protein. To this end, eukaryotic hostcells which possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483,Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that doesnot endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g.,COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W,L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells. In certainembodiments, anti-B7-H4 antibodies described herein (e.g., an antibodyor antigen-binding fragment thereof comprising the CDRs of 20502,20502.1, or 22213) are produced in mammalian cells, such as CHO cells.

In certain embodiments, anti-B7-H4 antibodies described herein (e.g., anantibody or antigen-binding fragment thereof comprising the CDRs of20502, 20502.1, or 22213) are produced in Potelligent® CHOK1SV cells.

In some embodiments, host cells are provided that comprise nucleic acidencoding a B7-H4 antibody or antigen-binding fragment thereof describedherein, wherein the host cell lacks a functionalalpha-1,6-fucosyltransferase gene (FUT8) gene. In some embodiments, thehost cell is a CHO cell.

Once an antibody or antigen-binding fragment thereof described hereinhas been produced by recombinant expression, it can be purified by anymethod known in the art for purification of an immunoglobulin molecule,for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins.Further, the antibodies or antigen-binding fragments thereof describedherein can be fused to heterologous polypeptide sequences describedherein or otherwise known in the art to facilitate purification.

In specific embodiments, an antibody or antigen-binding fragment thereofdescribed herein is isolated or purified. Generally, an isolatedantibody or antigen-binding fragment thereof is one that issubstantially free of other antibodies or antigen-binding fragmentsthereof with different antigenic specificities than the isolatedantibody or antigen-binding fragment thereof. For example, in aparticular embodiment, a preparation of an antibody or antigen-bindingfragment thereof described herein is substantially free of cellularmaterial and/or chemical precursors.

1.3 Pharmaceutical Compositions

Provided herein are compositions comprising an antibody orantigen-binding fragment thereof described herein having the desireddegree of purity in a physiologically acceptable carrier, excipient orstabilizer (Remington's Pharmaceutical Sciences (1990) Mack PublishingCo., Easton, Pa.). Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed.

In various embodiments, compositions comprising an anti-B7-H4 antibodyor antigen-binding fragment thereof are provided in formulations with apharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: TheScience and Practice of Pharmacy with Facts and Comparisons: DrugfactsPlus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms andDrug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004);Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed.,Pharmaceutical Press (2000)).

Pharmaceutical compositions described herein can be useful in blockingthe inhibitory activity of B7-H4 against T cells and/or inADCC-dependent depletion of B7-H4 expressing cells. Pharmaceuticalcompositions described herein can be useful in treating a condition suchas cancer. Examples of cancer that can be treated in accordance with themethods described herein include, but are not limited to, breast cancer(e.g., triple negative breast cancer, ductal carcinoma), endometrialcarcinoma, ovarian cancer, and non-small cell lung cancer (e.g.,squamous cell carcinoma), pancreatic cancer, thyroid cancer, kidneycancer (e.g., renal cell carcinoma), and bladder cancer (e.g.,urothelial cell carcinoma). A non-small cell lung cancer can be, e.g.,an adenocarcinoma. Additional examples of cancer that can be treated inaccordance with the methods described herein include, but are notlimited to, head and neck cancer, small cell lung cancer, gastriccancer, melanoma, and cholangiocarcinoma. In one embodiment, the ovariancancer is a serous adenocarcinoma. In one embodiment, the breast canceris a ductal adenocarcinoma.

The pharmaceutical compositions described herein are in one embodimentfor use as a medicament. The pharmaceutical compositions describedherein are in one embodiment for use as a diagnostic, e.g., to detectthe presence of B7-H4 in a sample obtained from a patient (e.g., a humanpatient).

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

In some embodiments, pharmaceutical compositions are provided, whereinthe pharmaceutical composition comprises anti-B7-H4 antibodies orantigen-binding fragments thereof described herein and apharmaceutically acceptable carrier. In some embodiments, pharmaceuticalcompositions are provided, wherein the pharmaceutical compositioncomprises afucosylated anti-B7-H4 antibodies or antigen-bindingfragments thereof described herein and a pharmaceutically acceptablecarrier. In specific embodiments, such pharmaceutical compositioncomprises afucosylated anti-B7-H4 antibodies or antigen-bindingfragments e.g., wherein at least 80% of the antibodies in thecomposition are afucosylated. In specific embodiments, suchpharmaceutical composition comprises afucosylated anti-B7-H4 antibodiesor antigen-binding fragments e.g., wherein at least 85% of theantibodies in the composition are afucosylated. In specific embodiments,such pharmaceutical composition comprises afucosylated anti-B7-H4antibodies or antigen-binding fragments e.g., wherein at least 90% ofthe antibodies in the composition are afucosylated. In specificembodiments, such pharmaceutical composition comprises afucosylatedanti-B7-H4 antibodies or antigen-binding fragments e.g., wherein atleast 95% of the antibodies in the composition are afucosylated. Inspecific embodiments, such pharmaceutical composition comprisesafucosylated anti-B7-H4 antibodies or antigen-binding fragments e.g.,wherein at least 96% of the antibodies in the composition areafucosylated. In specific embodiments, such pharmaceutical compositioncomprises afucosylated anti-B7-H4 antibodies or antigen-bindingfragments e.g., wherein at least 97% of the antibodies in thecomposition are afucosylated. In specific embodiments, suchpharmaceutical composition comprises afucosylated anti-B7-H4 antibodiesor antigen-binding fragments e.g., wherein at least 98% of theantibodies in the composition are afucosylated. In specific embodiments,such pharmaceutical composition comprises afucosylated anti-B7-H4antibodies or antigen-binding fragments e.g., wherein at least 99% ofthe antibodies in the composition are afucosylated. In specificembodiments, such pharmaceutical composition comprises afucosylatedanti-B7-H4 antibodies or antigen-binding fragments wherein fucose isundetectable in the composition.

In some embodiments, a pharmaceutical composition comprises (i) anisolated antibody or antigen-binding fragment thereof that specificallybinds to human B7-H4, comprising (a) the heavy chain variable region(VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 andlight chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQID NOs:458-463, respectively, (b) a variable heavy chain regioncomprising the amino acid sequence of SEQ ID NO:464 and a variable lightchain region comprising the amino acid sequence of SEQ ID NO:42, or (c)a heavy chain comprising the amino acid sequence of SEQ ID NO:469 and alight chain comprising the amino acid sequence of SEQ ID NO:44, and (ii)a pharmaceutically acceptable excipient.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments thereof that specifically bindto human B7-H4 and comprise the heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ IDNOs:458-463, respectively and (ii) a pharmaceutically acceptableexcipient, wherein at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% of theantibodies or antigen-binding fragments thereof in the composition areafucosylated. In one embodiment, (i) the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region comprising theamino acid sequence of SEQ ID NO:464 and a variable light chain regioncomprising the amino acid sequence of SEQ ID NO:42 or (ii) the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:469 and a light chain comprising the amino acid sequence of SEQ IDNO:44.

In some embodiments, a pharmaceutical composition comprises (i) anisolated antibody or antigen-binding fragment thereof that specificallybinds to human B7-H4, comprising (a) the heavy chain variable region(VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 andlight chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQID NOs:35-40, respectively, (b) a variable heavy chain region comprisingthe amino acid sequence of SEQ ID NO:41 and a variable light chainregion comprising the amino acid sequence of SEQ ID NO:42, or (c) aheavy chain comprising the amino acid sequence of SEQ ID NO:43 and alight chain comprising the amino acid sequence of SEQ ID NO:44, and (ii)a pharmaceutically acceptable excipient.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments thereof that specifically bindto human B7-H4 and comprise the heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ IDNOs:35-40, respectively and (ii) a pharmaceutically acceptableexcipient, wherein at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% of theantibodies or antigen-binding fragments thereof in the composition areafucosylated. In one embodiment, (i) the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region comprising theamino acid sequence of SEQ ID NO:41 and a variable light chain regioncomprising the amino acid sequence of SEQ ID NO:42 or (ii) the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:43 and a light chain comprising the amino acid sequence of SEQ IDNO:44.

In some embodiments, a pharmaceutical composition comprises (i) anisolated antibody or antigen-binding fragment thereof that specificallybinds to human B7-H4, comprising (a) the heavy chain variable region(VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 andlight chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQID NOs:65-70, respectively, (b) a variable heavy chain region comprisingthe amino acid sequence of SEQ ID NO:71 and a variable light chainregion comprising the amino acid sequence of SEQ ID NO:72, or (c) aheavy chain comprising the amino acid sequence of SEQ ID NO:73 and alight chain comprising the amino acid sequence of SEQ ID NO:74, and (ii)a pharmaceutically acceptable excipient.

Also provided herein is a pharmaceutical composition comprising (i)antibodies or antigen-binding fragments thereof that specifically bindto human B7-H4 and comprise the heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ IDNOs:65-70, respectively and (ii) a pharmaceutically acceptableexcipient, wherein at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% of theantibodies or antigen-binding fragments thereof in the composition areafucosylated. In one embodiment, (i) the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region comprising theamino acid sequence of SEQ ID NO:71 and a variable light chain regioncomprising the amino acid sequence of SEQ ID NO:72 or (ii) the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:73 and a light chain comprising the amino acid sequence of SEQ IDNO:74.

1.4 Uses and Methods

1.4.1 Therapeutic Uses and Methods

In one aspect, presented herein are methods for modulating one or moreimmune functions in a subject, comprising administering to a subject inneed thereof an anti-B7-H4 antibody or antigen-binding fragment thereofdescribed herein, or a pharmaceutical composition thereof as describedabove and herein.

In another embodiment, an anti-B7-H4 antibody or antigen-bindingfragment thereof, or pharmaceutical composition, is administered to apatient (e.g., a human patient) to increase the proliferation of Tcells, CD4+ T cells, or CD8+ T cells in the patient. In anotherembodiment, an anti-B7-H4 antibody or antigen-binding fragment thereof,or pharmaceutical composition, is administered to a patient (e.g., ahuman patient) to increase interferon-gamma (IFNγ) production in thepatient. In another embodiment, an anti-B7-H4 antibody orantigen-binding fragment thereof, or pharmaceutical composition, isadministered to a patient (e.g., a human patient) to block theinhibitory activity of B7-H4 against T cells in the patient. In anotherembodiment, an anti-B7-H4 antibody or antigen-binding fragment thereof,or pharmaceutical composition, is administered to a patient (e.g., ahuman patient) to deplete B7-H4 expressing cancer cells in the patient.In another embodiment, the anti-B7-H4 antibody or antigen-bindingfragment thereof, or pharmaceutical composition, is administered toachieve two or more of the above effects.

In a certain embodiment, provided herein are methods of treating acancer, e.g., a B7-H4 expressing cancer. The method of treating cancercan comprise administering an anti-B7-H4 antibody or antigen-bindingfragment thereof provided herein or a pharmaceutical compositioncomprising an anti-B7-H4 antibody or antigen-binding fragment thereofprovided herein to a patient (e.g., a human patient) in need thereof.

In a certain embodiment, provided herein are methods of treating acancer selected from the group consisting of: breast cancer (e.g.,triple negative breast cancer, ductal carcinoma), endometrial carcinoma,ovarian cancer, non-small cell lung cancer (e.g., squamous cellcarcinoma), pancreatic cancer, thyroid cancer, kidney cancer (e.g.,renal cell carcinoma), and bladder cancer (e.g., urothelial cellcarcinoma). In a certain embodiment, provided herein are methods oftreating a non-small cell lung cancer that is an adenocarcinoma. In acertain embodiment, provided herein are methods of treating a cancerselected from the group consisting of: head and neck cancer, small celllung cancer, gastric cancer, melanoma, and cholangiocarcinoma. In oneembodiment, the ovarian cancer is a serous adenocarcinoma. In oneembodiment, the breast cancer is a ductal adenocarcinoma. In someembodiments, such methods comprise administering an anti-B7-H4 antibodyor antigen-binding fragment thereof provided herein or a pharmaceuticalcomposition comprising an anti-B7-H4 antibody or antigen-bindingfragment thereof provided herein to a patient (e.g., a human patient) inneed thereof. In some embodiments, the cancer is a B7-H4 expressingcancer.

In a certain embodiment, provided herein is a method of treating acancer that is a PD-1/PD-L1 inhibitor inadequate responder. A cancerthat is a PD-1/PD-L1 inhibitor inadequate responder, may have previouslyresponded to a PD-1/PD-L1 inhibitor, but may have become less responsiveto the PD-1/PD-L1 inhibitor, or the cancer may have never responded tothe PD-1/PD-L1 inhibitor. Inadequate response to a PD-1/PD-L1 inhibitormeans that aspects of the cancer that would be expected to improvefollowing a standard dose of the PD-1/PD-L1 inhibitor do not improve,and/or improvement only occurs if greater than a standard dose isadministered. In some embodiments, a subject with a cancer that is aPD-1/PD-L1 inhibitor inadequate responder has experienced, or isexperiencing, an inadequate response to the PD-1/PD-L1 inhibitor afterreceiving a standard dose for at least two weeks, at least three weeks,at least four weeks, at least six weeks, or at least twelve weeks. A“standard” dose is determined by a medical professional, and may dependon the subject's age, weight, healthy history, severity of disease, thefrequency of dosing, etc. In some embodiments, subject with a cancerthat is a PD-1/PD-L1 inhibitor inadequate responder has experienced, oris experiencing, an inadequate response to an anti-PD-1 antibody and/oran anti-PD-L1 antibody. In some embodiments, a subject with cancer thatis a PD-1/PD-L1 inhibitor inadequate responder has experienced, or isexperiencing, an inadequate response to AMP-224. In some embodiments, asubject with cancer that is a PD-1/PD-L1 inhibitor inadequate responderhas experienced, or is experiencing, an inadequate response to aPD-1/PD-L1 inhibitor selected from nivolumab, pembrolizumab, andatezolizumab.

In a certain embodiment, provided herein is a method of treating acancer that expresses a low level of PD-L1. In some embodiments, acancer that expresses a “low level of PD-L1,” or expresses “PD-L1 at alow level,” denotes that the level of PD-L1 is under the level ofexpression for a cancer that is indicated for treatment with a PD-1 orPD-L1 antagonist in which patients are selected for treatment based onPD-L1 expression levels. In some embodiments, a “low level of PD-L1” isone in which less than 1% of the cells in the tumor have membranestaining. In some embodiments, a “low level” in regard to PD-L1 is lessthan 1% staining, for example, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2,0.1% or 0% of the cells of the tumor are stained. In some embodiments,PD-L1 expression levels can be measured by chromogenic IHC orimmunofluorescence IHC (Aqua scoring). In certain embodiments, PD-L1staining of 5% or less (including tumor and/or immune cells) canindicate that a sample expresses a “low level of PD-L1.” In certainembodiments, PD-L1 staining of 10% or less (including tumor and/orimmune cells) can indicate that a sample expresses a “low level ofPD-L1.” Unless indicated otherwise herein, a 5% threshold is used herein(i.e., 5% or less indicates a “low level of PD-L1”).

In another embodiment, an anti-B7-H4 antibody or antigen-bindingfragment thereof, or pharmaceutical composition, is administered to apatient (e.g., a human patient) diagnosed with cancer to increase theproliferation of T cells, CD4+ T cells, or CD8+ T cells in the patient.In another embodiment, an anti-B7-H4 antibody or antigen-bindingfragment thereof, or pharmaceutical composition, is administered to apatient (e.g., a human patient) diagnosed with cancer to increaseinterferon-gamma (IFNγ) production in the patient. In anotherembodiment, an anti-B7-H4 antibody or antigen-binding fragment thereof,or pharmaceutical composition, is administered to a patient (e.g., ahuman patient) diagnosed with cancer to block the inhibitory activity ofB7-H4 against T cells in the patient. In another embodiment, ananti-B7-H4 antibody or antigen-binding fragment thereof, orpharmaceutical composition, is administered to a patient (e.g., a humanpatient) diagnosed with cancer to deplete B7-H4 expressing cancer cellsin the patient.

In further embodiments, an anti-B7-H4 antibody or antigen-bindingfragment thereof, or pharmaceutical composition, is administered to apatient as provided above, and further in combination with an additionaltherapeutic agent, e.g., a chemotherapeutic agent or an immunestimulating agent, such as a T cell checkpoint inhibitor.

In an exemplary embodiment, the additional therapeutic agent is a PD-1antagonist, such as an antagonistic PD-1 antibody. Suitable PD-1antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA(pembrolizumab), or MEDI-0680 (AMP-514; WO2012/145493). Suitable PD-1antibodies also include, for example, camrelizumab (SHR-1210),tislelizumab (BGB-A317), or spartalizumab (NPVPDR001, NVS240118,PDR001). The additional therapeutic agent may also include pidilizumab(CT-011). A recombinant protein composed of the extracellular domain ofPD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224, can alsobe used to antagonize the PD-1 receptor.

In another exemplary embodiment, the additional therapeutic agent is aPD-L1 antagonist, such as an antagonistic PD-L1 antibody. Suitable PD-L1antibodies include, for example, TECENTRIQ (atezolizumab), durvalumab(MEDI4736), BMS-936559 (WO2007/005874), MSB0010718C (WO2013/79174) orrHigM12B7.

In yet another exemplary embodiment, the additional therapeutic agent isa GITR agonist, such as an agonistic GITR antibody. Suitable GITRantibodies include, for example, TRX-518 (WO06/105021, WO09/009116),MK-4166 (WO11/028683), or a GITR antibody disclosed in WO2015/031667. Inanother embodiment, the additional therapeutic agent is a GITR antibodydisclosed in WO2017/015623. In a particular embodiment, the GITRantibody is selected from: a) an antibody comprising a GITR bindingdomain (GITR-BD) comprising a CDR1 comprising the sequence of SEQ IDNO:411, a CDR2 comprising the sequence of SEQ ID NO:412, and a CDR3comprising the sequence of SEQ ID NO:413; b) an antibody comprising aGITR-BD comprising the sequence of SEQ ID NO:414; c) a tetravalentmolecule comprising two copies of a polypeptide having the structure(GITR-BD)-Linker-(GITR-BD)-Linker-Hinge-Fc, wherein (i) the GITR-BDcomprises a CDR1 comprising the sequence of SEQ ID NO:411, a CDR2comprising the sequence of SEQ ID NO:412, and a CDR3 comprising thesequence of SEQ ID NO:413, (ii) the Linker is a polypeptide, (iii) theHinge is a polypeptide derived from an immunoglobulin hinge region, and(iv) the Fc is an immunoglobulin Fc polypeptide; d) a tetravalentmolecule comprising two copies of a polypeptide having the structure(GITR-BD)-Linker-(GITR-BD)-Linker-Hinge-Fc, wherein (i) the GITR-BDcomprises the amino acid sequence of SEQ ID NO:414, (ii) the Linker is apolypeptide, (iii) the Hinge is a polypeptide derived from animmunoglobulin hinge region, and (iv) the Fc is an immunoglobulin Fcpolypeptide; and e) a tetravalent molecule comprising two copies of apolypeptide comprising the sequence of SEQ ID NO: 415. In any of theabove embodiments of a tetravalent molecule, a Hinge may comprise thesequence of SEQ ID NO:416, 417 or 418. In any of the above embodimentsof a tetravalent molecule, a Linker may comprise an amino acid sequenceselected from GG, GGG, and SEQ ID NOs:419-425. In certain embodiments,the Hinge comprises SEQ ID NO:417 and the Linker comprises any one ofSEQ ID NOs:419-423.

In yet another exemplary embodiment, the additional therapeutic agent isa CD80 extracellular domain (CD80 ECD), e.g., SEQ ID NO:426; or a CD80ECD fusion molecule comprising CD80 ECD and a fusion partner, asdisclosed in WO2017/079117. In certain embodiments, the CD80 ECD fusionmolecule is a CD80 ECD-Fc fusion protein. In a specific embodiment, theCD80 ECD-Fc fusion protein comprises the sequence of SEQ ID NO:427 or428.

In yet another exemplary embodiment, the additional therapeutic agent isan anti-CSF1R antibody disclosed in U.S. Pat. Nos. 8,182,813, 8,206,715,8,263,079, 8,513,199 or 9,221,910. In a particular embodiment, theanti-CSF1R antibody is selected from: a) an antibody comprising a heavychain comprising the sequence of SEQ ID NO:429 and a light chaincomprising the sequence of SEQ ID NO:430; b) an antibody comprising aheavy chain comprising a heavy chain (HC) complementarity determiningregion 1 (CDR1) comprising the sequence of SEQ ID NO:431, an HC CDR2comprising the sequence of SEQ ID NO:432, and an HC CDR3 comprising thesequence of SEQ ID NO:433, and a light chain comprising a light chain(LC) CDR1 comprising the sequence of SEQ ID NO:434, a LC CDR2 comprisingthe sequence of SEQ ID NO:435, and a LC CDR3 comprising the sequence ofSEQ ID NO:436; and c) an antibody comprising a heavy chain comprisingthe sequence of SEQ ID NO:437 and a light chain comprising the sequenceof SEQ ID NO:438.

Usually, the patient is a human but non-human mammals includingtransgenic mammals can also be treated.

In some embodiments, the present invention relates to an antibody orantigen-binding fragment thereof or pharmaceutical composition providedherein for use as a medicament. In some aspects, the present inventionrelates to an antibody or antigen-binding fragment thereof orpharmaceutical composition provided herein, for use in a method for thetreatment of cancer. In some aspects, the present invention relates toan antibody or antigen-binding fragment thereof or pharmaceuticalcomposition provided herein, for use in a method for the treatment ofcancer in a subject, comprising administering to the subject aneffective amount of an antibody or antigen-binding fragment thereof orpharmaceutical composition provided herein.

1.4.1.1 Routes of Administration & Dosage

An antibody or antigen-binding fragment thereof or composition describedherein can be delivered to a subject by a variety of routes, such asparenteral, subcutaneous, intravenous, intradermal, transdermal,intranasal, intratumoral, and administration to a tumor draining lymphnode. In one embodiment, the antibody or antigen-binding fragmentthereof or composition is administered by an intravenous route.

The amount of an antibody or antigen-binding fragment thereof orcomposition which will be effective in the treatment of a condition willdepend on the nature of the disease. The precise dose to be employed ina composition will also depend on the route of administration, and theseriousness of the disease.

1.4.2 Detection & Diagnostic Uses

An anti-B7-H4 antibody or antigen-binding fragment thereof describedherein (see, e.g., Section 5.2) can be used to assay B7-H4 proteinlevels in a biological sample using classical methods known to those ofskill in the art, including immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA), immunoprecipitation, or Western blotting.Suitable antibody assay labels are known in the art and include enzymelabels, such as, glucose oxidase; radioisotopes, such as iodine (125I,121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), andtechnetium (99Tc); luminescent labels, such as luminol; and fluorescentlabels, such as fluorescein and rhodamine, and biotin. Such labels canbe used to label an antibody or antigen-binding fragment thereofdescribed herein. Alternatively, a second antibody or antigen-bindingfragment thereof that recognizes an anti-B7-H4 antibody orantigen-binding fragment thereof described herein can be labeled andused in combination with an anti-B7-H4 antibody or antigen-bindingfragment thereof to detect B7-H4 protein levels.

Assaying for the expression level of B7-H4 protein is intended toinclude qualitatively or quantitatively measuring or estimating thelevel of a B7-H4 protein in a first biological sample either directly(e.g., by determining or estimating absolute protein level) orrelatively (e.g., by comparing to the disease associated protein levelin a second biological sample). B7-H4 polypeptide expression level inthe first biological sample can be measured or estimated and compared toa standard B7-H4 protein level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” B7-H4 polypeptide level is known, it can be used repeatedlyas a standard for comparison.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing B7-H4. Methods for obtaining tissuebiopsies and body fluids from animals (e.g., humans) are well known inthe art. Biological samples include peripheral mononuclear blood cells.A biological sample may also be a blood sample, in which circulatingtumor cells (or “CTCs”) may express B7-H4 and be detected.

An anti-B7-H4 antibody described herein can be used for prognostic,diagnostic, monitoring and screening applications, including in vitroand in vivo applications well known and standard to the skilled artisanand based on the present description. Prognostic, diagnostic, monitoringand screening assays and kits for in vitro assessment and evaluation ofimmune system status and/or immune response may be utilized to predict,diagnose and monitor to evaluate patient samples including those knownto have or suspected of having an immune system-dysfunction or cancer.This type of prognostic and diagnostic monitoring and assessment isalready in practice utilizing antibodies against the HER2 protein inbreast cancer (HercepTest™, Dako) where the assay is also used toevaluate patients for antibody therapy using Herceptin®. In vivoapplications include directed cell therapy and immune system modulationand radio imaging of immune responses.

Anti-B7-H4 antibodies and antigen-binding fragments thereof describedherein can carry a detectable or functional label. When fluorescencelabels are used, currently available microscopy andfluorescence-activated cell sorter analysis (FACS) or combination ofboth methods procedures known in the art may be utilized to identify andto quantitate the specific binding members. Anti-B7-H4 antibodies orantigen-binding fragments thereof described herein can carry afluorescence label. Exemplary fluorescence labels include, for example,reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein andTexas red, Alexa Fluor dyes, Cy dyes and DyLight dyes. An anti-B7-H4antibody can carry a radioactive label, such as the isotopes 3H, 14C,32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 67Cu, 90Y, 99Tc, 111In, 117Lu,121I, 124I, 125I, 131I, 198Au, 211At, 213Bi, 225Ac and 186Re. Whenradioactive labels are used, currently available counting proceduresknown in the art may be utilized to identify and quantitate the specificbinding of anti-B7-H4 antibody or antigen-binding fragment to B7-H4(e.g., human B7-H4). In the instance where the label is an enzyme,detection may be accomplished by any of the presently utilizedcolorimetric, spectrophotometric, fluorospectrophotometric, amperometricor gasometric techniques as known in the art. This can be achieved bycontacting a sample or a control sample with an anti-B7-H4 antibody orantigen-binding fragment thereof under conditions that allow for theformation of a complex between the antibody or antigen-binding fragmentthereof and B7-H4. Any complexes formed between the antibody orantigen-binding fragment thereof and B7-H4 are detected and compared inthe sample and the control. In light of the specific binding of theantibodies or antigen-binding fragments thereof described herein forB7-H4, the antibodies or antigen-binding fragments thereof can be usedto specifically detect B7-H4 expression on the surface of cells. Theantibodies or antigen-binding fragments thereof described herein canalso be used to purify B7-H4 via immunoaffinity purification.

Also included herein is an assay system which may be prepared in theform of a test kit for the quantitative analysis of the extent of thepresence of, for instance, B7-H4. The system or test kit may comprise alabeled component, e.g., a labeled antibody or antigen-binding fragment,and one or more additional immunochemical reagents. See, e.g., Section5.6 below for more on kits.

In some aspects, methods for in vitro detecting B7-H4 in a sample,comprising contacting said sample with an antibody or antigen-bindingfragment thereof, are provided herein. In some aspects, provided hereinis the use of an antibody or antigen-binding fragment thereof providedherein, for in vitro detecting B7-H4 in a sample. In one aspect,provided herein is an antibody or antigen-binding fragment thereof orpharmaceutical composition provided herein for use in the detection ofB7-H4 in a subject or a sample obtained from a subject. In one aspect,provided herein is an antibody or antigen-binding fragment thereof orpharmaceutical composition provided herein for use as a diagnostic. Inone preferred embodiment, the antibody comprises a detectable label. Inone preferred embodiment, B7-H4 is human B7-H4. In one preferredembodiment, the subject is a human.

1.5 Kits

Provided herein are kits comprising one or more antibodies orantigen-binding fragments thereof described herein or conjugatesthereof. In a specific embodiment, provided herein is a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions described herein,such as one or more antibodies or antigen-binding fragments thereofprovided herein. Optionally associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

Also provided herein are kits that can be used in diagnostic methods. Inone embodiment, a kit comprises an antibody or antigen-binding fragmentthereof described herein, preferably a purified antibody orantigen-binding fragment thereof, in one or more containers. In aspecific embodiment, kits described herein contain a substantiallyisolated B7-H4 antigen (e.g., human B7-H4) that can be used as acontrol. In another specific embodiment, the kits described hereinfurther comprise a control antibody or antigen-binding fragment thereofwhich does not react with a B7-H4 antigen. In another specificembodiment, kits described herein contain one or more elements fordetecting the binding of an antibody or antigen-binding fragment thereofto a B7-H4 antigen (e.g., the antibody or antigen-binding fragmentthereof can be conjugated to a detectable substrate such as afluorescent compound, an enzymatic substrate, a radioactive compound ora luminescent compound, or a second antibody or antigen-binding fragmentthereof which recognizes the first antibody or antigen-binding fragmentthereof can be conjugated to a detectable substrate). In specificembodiments, a kit provided herein can include a recombinantly producedor chemically synthesized B7-H4 antigen. The B7-H4 antigen provided inthe kit can also be attached to a solid support. In a more specificembodiment, the detecting means of the above described kit includes asolid support to which a B7-H4 antigen is attached. Such a kit can alsoinclude a non-attached reporter-labeled anti-human antibody orantigen-binding fragment thereof or anti-mouse/rat antibody orantigen-binding fragment thereof. In this embodiment, binding of theantibody or antigen-binding fragment thereof to the B7-H4 antigen can bedetected by binding of the said reporter-labeled antibody orantigen-binding fragment thereof.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

The examples in this Section (i.e., Section 6) are offered by way ofillustration, and not by way of limitation.

Example 1 Assessment of Prevalence of B7-H4 Expression in MultipleIndications

The B7-H4 mouse monoclonal antibody A57.1 (ATCC Catalog No. PTA-5180)was used to detect the presence of B7-H4 on archival samples, a mixtureof whole sections, and tumor microarrays. The samples were treated withthe primary antibody and detected using a polymer detection systemattached to DAB (Ventana Medical Systems).

B7-H4 was readily detected in the membrane and the cytosol in tumortissues harvested from a variety of cancer patients, including invasiveductal carcinoma, triple negative breast cancer, ovarian cancer,non-small cell lung cancer and endometrial cancer. (FIG. 1). Moreover,frequency of expression was also high in the indications listed in Table11.

TABLE 11 B7-H4 detection in tumors Tumor Type #Total #Positive PercentPositive Triple Negative Breast Cancer 74 58 78% Invasive DuctalCarcinoma 51 38 74.50%   Endometrial Carcinoma 77 54 70% Ovarian Cancer141 85 60% Non-Small Cell Lung Cancer 47 19 40% (Squamous)

B7-H4 is expressed in other cancers, such as kidney cancer (e.g., renalcell carcinoma), bladder cancer (e.g., urothelial cell carcinoma),pancreatic cancer, and thyroid cancer. See e.g., Zhu, J., et al., AsianPacific J. Cancer Prev. 14: 3011-3015 (2011), Krambeck A, et al., PNAS103: 10391-10396 (2006), Fan, M. et al., Int. J. Clin. Exp. Pathol. 7:6768-6775 (2014), Xu, H., et al., Oncology Letters 11: 1841-1846 (2016),and Liu, W., et al., Oncology Letters 8: 2527-2534 (2014).

In a subsequent experiment, B7-H4 prevalence in various tumor types wasassessed by immunohistochemistry (IHC) (FIG. 1B). The following tumortypes and stages of disease were tested: endometrial, breast invasiveductal carcinoma (IDC), breast triple negative (TN), breast triplenegative (late stage), ovarian cancer, bladder cancer (early stage),non-small cell lung cancer (sq (squamous), early stage), non-small celllung cancer (sq, late stage), head and neck cancer, bladder (latestage), small cell lung cancer, cholangiocarcinoma, non-small cell lungcancer (ad (adenocarcinoma)), thyroid cancer, pancreatic cancer, gastriccancer, melanoma, glioblastoma or glioblastoma multiforme (GBM), andmerkel cell carcinoma. B7-H4 surface protein expression on tumor cellswas evaluated on procured tumor samples from various tumor types andstages of disease where available (early stage (I/II), late stage(III/IV)). IHC intensity with the antibody A57.1 was scored by category0 to 3, and samples were attributed a single IHC score by the highestintensity observed in a minimum of 10% of all tumor cells per wholesection. A total of 50 to 100 samples per tumor type were evaluated.

Example 2 Generation of Novel Antibodies Against Human B7-H4

B7-H4-specific antibodies were isolated from full-length human IgG1naïve antibody libraries using an in vitro yeast presentation system.The libraries are designed to mimic the immune system; they do notcontain pre-defined heavy and light chain pairs. Libraries weresubjected to multiple rounds of positive and negative selectionstrategies using B7-H4 protein to enrich for IgG that werecross-reactive to human, cynomolgus, and murine B7-H4 target. After fourrounds of selection, the resulting IgG were sequenced, and uniqueantibodies were produced and evaluated for both avid and monomericbinding affinity to recombinant B7-H4 ectodomain, for epitope binning,and for target-specific cell binding.

A more detailed description of the selection, affinity maturation, andanalytical methods that were used to generate and characterize the B7-H4antibodies is provided below.

Materials and Methods

Antigens were biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kitfrom Pierce. Goat F(ab′)2 anti-human kappa-FITC (LC-FITC), ExtrAvidin-PE(EA-PE) and Streptavidin-AF633 (SA-633) were obtained from SouthernBiotech, Sigma, and Molecular Probes, respectively. StreptavidinMicroBeads and MACS LC separation columns were purchased from MiltenyiBiotec. Goat anti-human IgG-PE Human-PE) was obtained from SouthernBiotech.

Naïve Discovery

Eight naïve human synthetic yeast libraries each of ˜109 diversity werepropagated as previously described (see Y. Xu et al, Addressingpolyspecificity of antibodies selected from an in vitro yeastpresentation system: a FACS-based, high-throughput selection andanalytical tool. PEDS 26.10, 663-70 (2013); WO2009036379; WO2010105256;and WO2012009568.) For the first two rounds of selection, a magneticbead sorting technique utilizing the Miltenyi MACS system was performed,as previously described (see Siegel et al, High efficiency recovery andepitope-specific sorting of an scFv yeast display library.” J ImmunolMethods 286(1-2), 141-153 (2004).) Briefly, yeast cells (˜1010cells/library) were incubated with 10 ml of 10 nM biotinylated Fc fusionantigen for 30 min at 30° C. in wash buffer (phosphate-buffered saline(PBS)/0.1% bovine serum albumin (BSA)). After washing once with 40 mlice-cold wash buffer, the cell pellet was resuspended in 20 mL washbuffer, and Streptavidin MicroBeads (500 μl) were added to the yeast andincubated for 15 min at 4° C. Next, the yeast were pelleted, resuspendedin 20 mL wash buffer, and loaded onto a Miltenyi LS column. After the 20mL were loaded, the column was washed 3 times with 3 ml wash buffer. Thecolumn was then removed from the magnetic field, and the yeast wereeluted with 5 mL of growth media and then grown overnight. The followingrounds of selection were performed using flow cytometry. Approximately2×107 yeast were pelleted, washed three times with wash buffer, andincubated at 30° C. with either decreasing concentrations ofbiotinylated Fc fusion antigen (10 to 1 nM) under equilibriumconditions, 10 nM biotinylated Fc fusion antigens of different speciesin order to obtain species cross-reactivity, or with a poly-specificitydepletion reagent (PSR) to remove non-specific antibodies from theselection. For the PSR depletion the libraries were incubated with a1:10 dilution of biotinylated PSR reagent as previously described (seeY. Xu et al., addressing polyspecificity of antibodies selected from anin vitro yeast presentation system: a FACS-based, high-throughputselection and analytical tool. PEDS 26.10, 663-70 (2013).) Yeast werethen washed twice with wash buffer and stained with LC-FITC (diluted1:100) and either SA-633 (diluted 1:500) or EAPE (diluted 1:50)secondary reagents for 15 min at 4° C. After washing twice with washbuffer, the cell pellets were resuspended in 0.3 mL wash buffer andtransferred to strainer-capped sort tubes. Sorting was performed using aFACS ARIA sorter (BD Biosciences) and sort gates were determined toselect for antibodies with desired characteristics. Selection roundswere repeated until a population with all of the desired characteristicswas obtained. After the final round of sorting, yeast were plated andindividual colonies were picked for characterization.

Antibody Optimization

Optimization of antibodies was performed via a light chain batch shufflemethod, and then by introducing diversities into the heavy chain andlight chain variable regions as described below. A combination of someof these approaches was used for each antibody.

Light chain batch shuffle: Heavy chain plasmids from a naïve selectionoutput were extracted from the yeast via smash and grab, propagated inand subsequently purified from E.coli, and transformed into a lightchain library with a diversity of 5×106. Selections were performed withone round of MACS and four rounds of FACS employing the same conditionsas the naïve discovery.

CDRH1 and CDRH2 selection: The CDRH3 of a single antibody was recombinedinto a premade library with CDRH1 and CDRH2 variants of a diversity of1×108 and selections were performed with one round of MACS and fourrounds of FACS as described in the naïve discovery. For each FACS roundthe libraries were looked at for PSR binding, species cross-reactivity,and affinity pressure, and sorting was performed in order to obtain apopulation with the desired characteristics. For these selectionsaffinity pressures were applied by using decreasing concentrations ofbiotinylated HIS-B7-H4 antigen (100 to 1 nM) under equilibriumconditions at 30° C.

VH Mut selection: The heavy chain variable region (VH) was mutagenizedvia error prone PCR. The library was then created by transforming thismutagenized VH and the heavy chain expression vector into yeast alreadycontaining the light chain plasmid of the parent. Selections wereperformed similar to previous cycles using FACS sorting for two rounds.For each FACS round the libraries were looked at for PSR binding andaffinity pressure, and sorting was performed in order to obtain apopulation with the desired characteristics. Affinity pressures forthese selections were performed as described above in the CDRH1 andCDRH2 selection.

CDRL1 and CDRL2 selection: The CDRL3 of a single antibody was recombinedinto a premade library with CDRL1 and CDRL2 variants of a diversity of˜5×105 and selections were performed similar to previous cycles usingFACS sorting for three rounds. For each FACS round the libraries werelooked at for PSR binding and affinity pressure, and sorting wasperformed in order to obtain a population with the desiredcharacteristics. Affinity pressures for these selections were performedas described above in the CDRH1 and CDRH2 selection.

Antibody Production and Purification

Yeast clones were grown to saturation and then induced for 48 h at 30°C. with shaking. After induction, yeast cells were pelleted and thesupernatants were harvested for purification. IgGs were purified using aProtein A column and eluted with acetic acid, pH 2.0. Fab fragments weregenerated by papain digestion and purified over KappaSelect (GEHealthcare LifeSciences).

ForteBio K_(D) Measurements

ForteBio affinity measurements were performed on an Octet RED384generally as previously described (see Estep et al, High throughputsolution-based measurement of antibody-antigen affinity and epitopebinning. Mabs 5(2), 270-278 (2013)). Briefly, ForteBio affinitymeasurements were performed by loading IgGs on-line onto AHQ sensors.Sensors were equilibrated off-line in assay buffer for 30 min and thenmonitored on-line for 60 seconds for baseline establishment. Sensorswith loaded IgGs were exposed to 100 nM antigen for 3 minutes, andafterwards were transferred to assay buffer for 3 min for off-ratemeasurement. For monovalent affinity assessment Fabs were used insteadof IgGs. For this assessment the unbiotinylated Fc fusion antigen wasloaded on-line onto the AHQ sensors. Sensors were equilibrated off-linein assay buffer for 30 min and then monitored on-line for 60 seconds forbaseline establishment. Sensors with loaded antigen were exposed to 100nM Fab for 3 minutes, and afterwards they were transferred to assaybuffer for 3 min for off-rate measurement. All kinetics were analyzedusing the 1:1 binding model.

ForteBio Epitope Binning/Ligand Blocking

Epitope binning/ligand blocking was performed using a standard sandwichformat cross-blocking assay. Control anti-target IgG was loaded onto AHQsensors and unoccupied Fc-binding sites on the sensor were blocked withan irrelevant human IgG1 antibody. The sensors were then exposed to 100nM target antigen followed by a second anti-target antibody or ligand.Additional binding by the second antibody or ligand after antigenassociation indicates an unoccupied epitope (non-competitor), while nobinding indicates epitope blocking (competitor or ligand blocking).

Four non-competing antibodies (“binning antibodies” 1, 2, 3, and 4) wereused to identify four distinct portions of the B7-H4 ectodomain by SPR.All of the antibodies generated during this campaign were then assessedfor competitive binding with these four binning antibodies to the B7-H4ectodomain. If a B7-H4 antibody competed with one of the four binningantibodies (e.g., binning antibody 1), the B7-H4 antibody was determinedto be in that BIN (e.g., BIN 1). If a B7-H4 antibody competed with twoof the four binning antibodies (e.g, binning antibodies 2 and 3), theB7-H4 antibody was determined to be in both of those BINS (e.g.,BIN2/3).

The IgG antibodies fell into at least four binding bins, with >90% ofthe antibodies binding to human/cyno or mouse B7-H4 with an affinityresponse ranging between >0.1 nm and <100 nM. Of the recombinant proteinbinders, approximately 75% also bind to B7-H4 on cells.

Cell Binding Analysis

100,000 cells overexpressing the antigen were washed with wash bufferand incubated with 100 ul 100 nM IgG for 5 minutes at room temperature.Cells were then washed twice with wash buffer and incubated with 100 ulof 1:100 anti-human IgGPE for 15 minutes on ice. Cells were then washedtwice with wash buffer and analyzed on a FACS Canto II analyzer (BDBiosciences.)

PSR Binding Assay

The PSR assay was done as previously described (see Xu Y, et al. (2013)Addressing polyspecificity of antibodies selected from an in vitro yeastpresentation system: A FACS-based, high-throughput selection andanalytical tool. Protein Eng Des Sel 26(10):663-670). In short, solublemembrane proteins were prepared from CHO cells. The enriched membranefraction was biotinylated using NHS-LCBiotin (Pierce, Thermo Fisher).This polyspecificity reagent was incubated with IgG-presenting yeast,followed by washing. Then secondary labeling mix (Extravidin-R-PE,anti-human LC-FITC, and propidium iodide) was added to the mixture.Samples were analyzed on a FACSCanto II analyzer (BD Biosciences) usingan HTS sample injector. Flow cytometry data were analyzed for meanfluorescence intensity (MFI) in the R-PE channel to assess nonspecificbinding. MFI values were normalized from 0 to 1 based on three referenceantibodies exhibiting low, medium, and high PSR MFI values.

Dynamic Scanning Fluorimetry

10 uL of 20× Sypro Orange is added to 20 uL of 0.2-1 mg/mL mAb or Fabsolution. A RT-PCR instrument (BioRad CFX96 RT PCR) is used to ramp thesample plate temperature from 40 to 95 C at 0.5 C increment, with 2 minequilibrate at each temperature. The negative of first derivative forthe raw data is used to extract Tm.

AC-SINS

The AC-SINS assay was performed as described previously (see Liu Y, etal. (2014) High-throughput screening for developability duringearly-stage antibody discovery using self-interaction nanoparticlespectroscopy. MAbs 6(2):483-492). In short, gold nanoparticles (TedPella Inc.) were coated with 80% capturing anti-human goat IgG Fc(Jackson ImmunoResearch) and 20% with polyclonal goat nonspecificantibody (Jackson ImmunoResearch). The antibodies of interest were thenincubated with the particles for 2 h and the wavelength shift wasmeasured using Molecular Devices SpectraMax M2 with SoftMax Pro6software. The self-interacting clones show a higher wavelength shiftaway from the PBS sample.

Example 3 Structural Characterization of Human Monoclonal AntibodiesAgainst B7-H4

The B7-H4 antibodies are human IgG1/kappa isotype. The variable heavy(VH) regions are comprised of alleles from the germline genes VH1, VH3,and VH4, and the variable light (VL) of alleles from germline genes thatinclude Vk1 and Vk3. Some subsets of the antibodies share high identitywithin the respective CDR regions of the VH and VL that can range from72-100%. Antibodies that share the same germline allele have highidentity in their VH and VL framework (FR) regions that ranges from88-100%. Sequences of the B7-H4 antibodies are provided in Tables 1-10.

Example 4 Characterization of Monoclonal Antibody Binding to B7-H4Protein

Binding affinities of anti-B7-H4 antibodies to B7-H4-extracellulardomain (ECD) were determined by biolayer interferometry (BLI). ForteBioaffinity assays are described in more detail above in Example 2.Briefly, the recombinant human B7-H4-huIgG1 (SEQ ID NO:409) protein wasimmobilized on Protein A tips, followed by an isotype control hIgG1 inorder to saturate any remaining binding sites on the capturing tip.Anti-B7-H4 antibodies were then evaluated for binding. In addition,antibody binding to cynomolgus monkey (SEQ ID NO: 2) and mouse B7-H4(SEQ ID NO: 3) was also assessed using the same protocol. The resultsare summarized in Table 12.

B7-H4-huIgG1: (SEQ ID NO: 409)MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE 12 B7-H4 Antibody Binding to Extracellular Domain by BiolayerInterferometry Human B7-H4 Cynomolgus B7-H4 Mouse B7-H4 Anti- K_(D)k_(on) k_(off) K_(D) k_(on) k_(off) K_(D) k_(on) k_(off) body BIN (M)(1/Ms) (1/s) (M) (1/Ms) (1/s) (M) (1/Ms) (1/s) 15441 2 7.15E−09 1.22E+068.72E−03 5.46E−08 1.33E+06 7.27E−02 NB — — 15461 2/3 6.29E−08 3.81E+052.40E−02 6.21E−08 4.34E+05 2.70E−02 5.57E−08 3.05E+05 1.70E−02 15462 2/3PF — — 6.65E−08 6.72E+05 4.47E−02 8.31E−08 4.59E+05 3.81E−02 15465 2/35.13E−08 4.84E+05 2.48E−02 5.11E−08 5.44E+05 2.78E−02 8.96E−08 2.96E+052.65E−02 15472 4 7.11E−09 7.00E+05 4.97E−03 7.80E−09 7.46E+05 5.82E−033.80E−08 4.22E+05 1.60E−02 15478 4 3.25E−08 4.77E+05 1.55E−02 3.11E−085.44E+05 1.69E−02 NB — — 15483 2/3 PF — — 1.08E−07 3.80E+05 4.09E−022.63E−07 2.65E+05 6.95E−02 15489 2/3 6.37E−08 3.66E+05 2.33E−02 7.21E−083.98E+05 2.87E−02 2.01E−07 2.93E+05 5.90E−02 15495 2 9.44E−09 2.89E+062.72E−02 5.17E−09 3.25E+06 1.68E−02 NB — — 15503 2 1.04E−08 1.96E+062.05E−02 6.32E−09 1.82E+06 1.15E−02 NB — — 20496 other 8.05E−10 6.89E+055.55E−04 7.21E−10 7.90E+05 5.69E−04 2.46E−08 6.92E+05 1.71E−02 20500 35.97E−09 3.91E+05 2.33E−03 5.47E−09 4.18E+05 2.29E−03 8.25E−09 4.08E+053.36E−03 20501 3 2.56E−09 9.80E+05 2.51E−03 2.42E−09 1.02E+06 2.46E−033.35E−09 1.12E+06 3.75E−03 20502 3 1.20E−09 9.36E+05 1.15E−03 1.20E−099.62E+05 1.12E−03 3.40E−09 6.32E+05 2.13E−03 20502.1 3 3.07E−09 4.87E+051.50E−03 2.64E−09 5.28E+05 1.39E−03 3.99E−09 5.20E+05 2.07E−03 20506 31.69E−09 4.41E+05 7.46E−04 1.48E−09 4.58E+05 6.78E−04 3.53E−09 4.66E+051.64E−03 20513 3 3.98E−09 4.53E+05 1.80E−03 3.67E−09 4.91E+05 1.80E−034.57E−09 5.17E+05 2.36E−03 20516 3 1.80E−08 4.10E+05 7.38E−03 1.76E−084.45E+05 7.83E−03 4.14E−08 3.98E+05 1.65E−02 22208 3 5.67E−09 1.84E+051.04E−03 5.44E−09 1.99E+05 1.08E−03 1.67E−08 9.95E+04 1.67E−03 22213 31.44E−08 4.96E+04 7.13E−04 7.48E−09 6.44E+04 4.82E−04 1.37E−08 4.03E+045.52E−04 22216 3 1.47E−09 2.45E+05 3.59E−04 1.47E−09 2.45E+05 3.59E−041.47E−09 2.45E+05 3.59E−04

In addition, the binding affinities of selected anti-B7-H4 antibodies tothe N-terminal domain of human B7-H4 (B7-H4 IgV-hulgG1; SEQ ID NO:410)were determined by surface plasmon resonance (SPR). Briefly, anti-humanFab antibody was immobilized on a carboxyl-derivatized SPR chip surface,and anti-B7-H4 antibodies were captured on the resulting surface at 5ug/ml for 30 seconds. B7-H4 IgV-hulgG1 at various concentrations (0 nM,3.7 nM, 11.1 nM, 33.3 nM, 100 nM, and 300 nM) was then flowed over thesurface and allowed to bind to the anti-B7-H4 antibodies during theassociation phase, followed by a buffer wash during the dissociationphase. Data was fitted using a 1:1 binding model, and the results aresummarized in Table 13.

B7-H4 IgV-huIgG1: (SEQ ID NO: 410)MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE 13 B7-H4 Antibody Binding to N-terminal Domain by Surface PlasmonResonance Human B7-H4 Antibody BIN K_(D) (M) k_(on) (1/Ms) k_(off) (1/s)15461 2/3 3.04E−08 1.71E+05 5.19E−03 15462 2/3 1.69E−08 2.95E+054.98E−03 15465 2/3 1.73E−08 2.41E+05 4.17E−03 15483 2/3 1.84E−082.41E+05 4.43E−03 15489 2/3 2.37E−08 1.64E+05 3.89E−03 20502 3 2.03E−091.78E+05 3.61E−04

Thus, multiple assays demonstrate that the antibodies bind to B7-H4.

Example 5 Epitope Mapping

Anti-B7-H4 antibody binding bins were determined by biolayerinterferometry (BLI). Epitope mapping assays are described in moredetail above in Example 2. Briefly, a first anti-B7-H4 antibody wascaptured on Protein A tips, followed by a control huIgG1 antibody tosaturate additional binding sites on the tips. Next, the sensor tipswere exposed to the antigen (B7-H4-huIgG1) (SEQ ID NO:409) followed bythe second anti-B7-H4 antibody, which was then evaluated for binding.Epitope bins were determined by comparison to binding after a non-B7-H4antibody was used as the first antibody. Results are summarized in Table12.

Example 6 Characterization of Monoclonal Antibody Binding to B7-H4Expressed on the Surface of Cells

HEK293T cell lines were transfected to express full-length human,cynomolgus monkey, mouse, or rat B7-H4. In addition, the endogenoushuman B7-H4 expressing SK-BR-3 breast cancer cell line was used toassess the ability of the antibodies to bind B7-H4 expressed on the cellsurface. 1×10⁵ parental HEK293T or transfected HEK293T cell lines wereincubated with 100 nM of the B7-H4 antibodies. Following incubation,cells were pelleted, washed, and incubated with a secondary labelingantibody, and samples were acquired on a flow cytometer. Fold bindingover the parental cell line (FOP) was calculated as follows: MFItransfected HKE293T cells/MFI parental HEK293T cells. A FOP valuegreater than 10 was considered to demonstrate specific binding.

To assess the cell binding potency of the B7-H4 antibodies, 1×105SK-BR-3 cells or 293 cells transfected to express cynomolgus monkey,mouse or rat B7-H4 were incubated with titrating doses of the B7-H4antibodies. Following incubation, cells were pelleted, washed andincubated with a secondary labeling antibody and samples were acquiredon a flow cytometer. Data was analyzed using the FlowJo software and MFIwas plotted vs. antibody concentration. The EC50 cell binding potencywas calculated using nonlinear regression curve fit (GraphPad Prism).

All B7-H4 antibodies demonstrated binding to HEK293T cells thatexpressed human B7-H4, cynomolgus monkey B7-H4, or mouse B7-H4.Antibodies also demonstrated potent dose-dependent binding to SK-BR-3cells endogenously expressing human B7-H4 on the cell surface or HEK293Tcell lines transfected to express cynomolgus monkey, mouse, or rat B7-H4on the cell surface. These data demonstrate that the majority of B7-H4antibodies are fully cross-reactive to cynomolgus monkey, mouse, and ratB7-H4 (FIGS. 2A and 2B, Table 14).

TABLE 14 B7-H4 Antibody Binding to Cell-Surface B7-H4 293- 293- 293-293- 293- 293- SK-BR-3 cynoB7- mouseB7- ratB7- huB7-H4 cynoB7-H4 moB7-H4(EC50; H4 (EC50; H4 (EC50; H4 (EC50; Antibody BIN (FOP) (FOP) (FOP) nM)nM) nM) nM) 15441 2 67 9 3 34.29 ND ND ND 15461 2/3 333 610 887 5.666.77 6.37 1.67 15462 2/3 530 416 456 4.55 5.29 5.40 1.13 15465 2/3 6261324 690 2.13 4.90 5.34 1.47 15472 4 580 1279 685 8.86 — — — 15478 4 5181130 125 PF — — — 15483 2/3 805 1012 1402 14.81 17.4  8.82 1.87 154892/3 612 1118 674 31.73 13.7  17.5  4.55 15495 2 619 524 6 61.2 — — —15503 2 289 877 5 PF — — — 20496 other 179 758 604 7.60 7.91 6.70 ND20500 3 182 630 478 1.54 3.38 4.00 1.14 20501 3 205 728 546 1.90 3.894.67 1.15 20502.1 3 200 683 559 2.50 4.94 5.57 1.26

Example 7 Characterization of Monoclonal Antibody T Cell CheckpointBlockade Activity

In order to characterize the T cell checkpoint blockade activity of theB7-H4 antibodies, primary human T cells were enriched from peripheralblood mononuclear cells (PBMCs) using the EasySep™ Human T CellEnrichment Kit based on manufacturer's instructions. Enriched T cellswere incubated at 2×10⁵ cell/mL with anti-CD3/anti-CD28 Dynabeads, at aone bead per cell ratio, at 37° C. Six days later, the beads weremagnetically removed, and the T cells were washed and incubated at 1×10⁶cell/mL with 10 U/mL IL-2 at 37° C. Four days later, T cells were washedand incubated at a 1×10⁶ cells/mL along with artificial antigenpresenting cells (aAPCs) at 2×10⁶ cells/mL at 37° C. in the presence of10 ug/mL of antibody or an antibody dose titration. aAPCs are a HEK293Tcell line that has been transfected to co-express a scFv format ofanti-human CD3 clone OKT3 and full-length human B7-H4 on the cellsurface. aAPCs were treated with Mitomycin C for one hour at 37° C. andthen thoroughly washed prior to adding to the T cell co-culture. 72hours after co-culture of T cells, aAPCs and B7-H4 antibodies, plateswere centrifuged, supernatants harvested and assessed for IFNγproduction by ELISA, and cells were harvested and stained to assessproliferation by FACS (specifically, the cells in each well wereincubated with antibodies against CD4 and CD8). Cells were then washed,fixed, permeabilized, and incubated with the Edu-Click reagent accordingthe manufacturer's instructions. Cells were washed, and the number ofproliferating CD4+, CD8+ or total T cells was measured using flowcytometry. Data was analyzed using the FlowJo software. For samples onlytreated with a single concentration of antibody, the data was calculatedand reported as a fold increase over control-huIgG treated samples. Forsamples treated with a dose titration of antibody, IFNγ production wasplotted vs. antibody concentration and the EC50 potency was calculatedusing nonlinear regression curve fit (GraphPad Prism).

All BIN2/3 and BIN3 antibodies reproducibly resulted in T cellcheckpoint blockade activity as measured by an increase in IFNγproduction and/or CD4⁺, CD8⁺ or total T cell proliferation. FIG. 3Ashows that antibody 15441 increases T cell proliferation by at least 3%and increases CD4+ and CD8+ T cell proliferation by at least 2%. FIG. 3Aalso shows that antibody 15461 increases T cell proliferation by atleast 21%, increases CD4+ T cell proliferation by at least 9%, andincreases CD8+ T cell proliferation by at least 11%. FIGS. 3B-3D showother dose-dependent T cell checkpoint blockade activities.

TABLE 15 B7-H4 Antibody - T Cell Checkpoint Blockade Activity aAPC AssayDonor 1 Donor 2 Donor 3 Donor 4 (EC50 +/− STD; (Max IFNγ, (Max IFNγ,(Max IFNγ, (Max IFNγ, Antibody BIN nM) pg/mL) pg/mL) pg/mL) pg/mL) 154612/3  531.18 +/− 403.66 311.64 164.66 1957.34 986.73 15462 2/3 1312.01+/− 1112.5 205.72 176.33 1661.59 484.8 15465 2/3 4310.72 +/− 7642.6230.45 133.64 1523.82 865.22 15483 2/3 728.84 +/− 711.1 93.2 87.841382.03 939.04 15489 2/3  291.31 +/− 327.82 82.47 96.63 1133.02 799.9

TABLE 16 B7-H4 Antibody Binding to Cell-Surface B7-H4 aAPC Assay Donor 1Donor 2 Donor 3 Donor 4 (EC50 +/− (Max IFNγ, (Max IFNγ, (Max IFNγ, (MaxIFNγ, Antibody BIN SID; nM) pg/mL) pg/mL) pg/mL) pg/mL) 20496 other PF185.88 ND ND ND 20500 3 0.84 +/− 0.35 821 273.83 117.9 4027.1 20501 30.79 +/− 0.18 849.6 310 135.6 4493.1 20502.1 3 0.79 +/− 0.03 937 303.9174.02 4269.7 20506 3 1.25 +/− 0.26 980.8 258.8 86.1 2301.8 20513 3 1.16+/− 0.16 658.8 240.3 91.1 1634.5 20516 3 PF ND 441.1 ND ND 22213 3 1.79+/− 1.83 ND ND ND ND

Example 8 Generation of Afucosylated and Fucosylated MonoclonalAntibodies

Antibodies with Fc regions having reduced fucose content in glycanmoieties may exhibit higher ADCC activity compared to a fullyfucosylated antibody (Niwa R et al., Clinical Cancer Research11(6):2327-36 (2005)). B7-H4 antibodies were generated in CHO-x cells(Yamane-Ohnuki N, et al. Biotechnology and Bioengineering 87(5): 614-22(2004)) to produce normally fucosylated antibodies and in a CHO cellline engineered to produce afucosylated antibodies (CHO-y cells) (id.).

Example 9 Characterization of Binding of Afucosylated and FucosylatedMonoclonal Antibodies

The fucosylated and afucosylated anti B7-H4 antibodies werecharacterized by SPR following protocol as described in Example 4. Theantibodies showed similar binding to human B7-H4 protein and thus thereis no impact of the glycosylation on binding (Table 17).

TABLE 17 Binding of afucosylated and fucosylated antibodies 293- 293-293- Human B7-H4 SK-BR-3 cynoB7-H4 mouseB7-H4 ratB7-H4 Antibody BINK_(D) (M) k_(on) (1/Ms) k_(off) (l/s) (EC50; nM) (EC50; nM) (EC50; nM)(EC50; nM) 20502 3 Fucosylated 2.58E−09 4.28E+05 1.10E−03 0.571 3.2914.371 2.486 20502 3 Afucosylated 2.74E−09 4.19E+05 1.15E−03 0.483 2.9231.655 2.446 20506 3 Fucosylated 1.93E−09 3.65E+05 7.03E−04 0.739 1.7701.77 3.249 20506 3 Afucosylated 2.01E−09 3.60E+05 7.25E−04 0.767 1.7691.769 PF 22213 3 Fucosylated 5.81E−09 1.53E+05 8.88E−04 0.651 5.1992.445 4.401 22213 3 Afucosylated 5.11E−09 1.69E+05 8.62E−04 0.667 3.8782.453 3.545

The fucosylated and afucosylated anti B7-H4 antibodies were alsocharacterized by flow cytometry. In these experiments, HEK293T celllines were transfected to express full-length cynomolgus monkey, mouse,or rat B7-H4. In addition, the endogenous human B7-H4 expressing SK-BR-3breast cancer cell lines were used to assess the cell binding potency ofB7-H4 antibodies. 1×105 SK-BR-3 cells or 293 cells transfected toexpress full-length human, cynomolgus monkey, mouse, or rat B7-H4 wereincubated with titrating doses of the B7-H4 antibodies. Followingincubation, cells were pelleted, washed, incubated with a secondarylabeling antibody, and run on a flow cytometer. Data were analyzed usingthe FlowJo software. MFI was plotted vs. antibody concentration, and theEC50 cell binding potency was calculated using nonlinear regressioncurve fit (GraphPad Prism).

The B7-H4 antibodies demonstrated potent dose-dependent binding toSK-BR-3 cells endogenously expressing B7-H4 on the cell surface or 293cell lines transfected to express cynomolgus monkey, mouse, or rat B7-H4on the cell surface. Binding potency and species cross-reactivity wasnot impacted by whether an antibody was fucosylated or afucosylated(FIGS. 4A-4D and Table 17).

Example 10 Binding of Afucosylated and Fucosylated Monoclonal Antibodiesto Human Fcγ Receptor IIIa (FcγRIIIa) V158 Allele

Antibody 20502 was produced as both fucosylated (Ab-F) and afucosylated(Ab-A) and tested for the binding affinity of the Fc region to FcgRIIIa(V158) by surface plasmon resonance (SPR). Briefly, Protein A wascovalently attached to a dextran chip using the amine coupling kit with100 mM ethylenediamine in 100 mM Sodium Borate buffer, pH 8.0 as theblocking reagent. Ab-A or Ab-F was captured at 2 densities on separateflow cells, and a Protein A derivatized flow served as a referencecontrol. Fc gamma RIIIA (V158) was diluted in HBS-P+ running buffer andinjected at 6 concentrations (0 nM, 1.37 nM, 12.3 nM, 37 nM, 111 nM, 333nM, and 1000 nM) in duplicate. The association constant, dissociationconstant, and affinity for Ab-A binding were calculated using theBiacore T200 Evaluation Software 1:1 binding model. The affinityconstant for Ab-A and Ab-F binding were determined using the BiacoreT200 Evaluation Software steady state affinity model. The afucosylatedB7-H4 antibody (Ab-A) has a 140-fold higher affinity for Fc gammareceptor IIIA (V158) than the same antibody with a fucosylated Fc (Ab-F)(FIGS. 5A and 5B, Table 18).

TABLE 18 Fcγ receptor IIIa (FcγRIIIa) V158 allele binding ka (1/Ms) kd(1/s) K_(D) (nM) Ab-A 6.46E+05 9.54E−10 15 Ab-F N/A N/A 210

Example 11 Afucosylated and Fucosylated Monoclonal Antibodies T CellCheckpoint Blockade Activity

Primary human T cells were enriched from PBMCs using the EasySep™ HumanT Cell Enrichment Kit based on the manufacturer's instructions. EnrichedT cells were incubated at 2×10⁵ cell/mL with anti-CD³/anti-CD28Dynabeads, at a one bead per cell ratio, at 37° C. Six days later, thebeads were magnetically removed, and T cells were washed and incubatedat 1×10⁶ cell/mL with 10 U/mL IL-2 at 37° C. Four days later, T cellswere washed and incubated at 1×10⁶ cells/mL along with artificialantigen presenting cells (aAPCs) at a 2×10⁶ cells/mL concentration at37° C. in the presence of B7-H4 antibody dose titration. aAPCs weretreated with Mitomycin C for one hour at 37° C. and then thoroughlywashed prior to adding to the T cell co-culture. 72 hours afterco-culture of T cells, aAPCs, and B7-H4 antibodies, plates werecentrifuged and supernatants were harvested and assessed for IFNγproduction by ELISA. IFNγ production was plotted vs. antibodyconcentration and the EC50 potency was calculated using nonlinearregression curve fit (GraphPad Prism).

The B7-H4 antibodies demonstrated potent T cell checkpoint blockadeactivity as measured by an increase in IFNγ production. Moreover, therewas no demonstrable difference in potency between afucosylated andfucosylated antibodies (FIG. 6A and Table 19.)

TABLE 19 T Cell Checkpoint blockade potency aAPC Assay (EC50 +/− STD;nM) Antibody BIN Afucosylated Fucosylated 20502 3 0.89 +/− 0.44 0.74 +/−0.39 20506 3 1.05 +/− 0.28 0.95 +/− 0.46 22213 3 0.16 +/− 0.05 0.17 +/−0.08

Next, primary human T cells were enriched from HLA-A2+ donor PBMCs usingthe Human Pan T Cell Isolation Kit following the manufacturer'sinstructions. MART-I TCR expressing T cells were generated by firstactivating 5×106 enriched Pan T cells with 1.5×107 anti-CD3/anti-CD28Dynabeads, 100 ng/mL IL-2 and 15 ng/mL IL-7 for 48 hours. 5×106activated T cells were then transduced with MART-I TCR lentiviralparticles in the presence of 200 ng/mL IL-2, 30 ng/mL IL-7 and 10 ug/mLpolybrene. 24 hours post transduction, MART-I TCR+ Pan T cells wereexpanded over a 15-day period in the presence of 33.3 ng/mL IL-2 and 5ng/mL IL-7. To generate HLA-A2 expressing target cell lines, theendogenous B7-H4 expressing human breast cancer cell lines MDA-MB-468and SK-BR-3 were transduced with HLA-A2 lentiviral particles for 48hours. Furthermore, B7-H4 was knocked-out of the HLA-A2+SK-BR-3 cellline. MART-I TCR+ Pan T cells were co-cultured in the presence of thevarious target cell lines at a 1:1 E:T ratio, 500 μg/mL of MART-Ipeptide and 67 nM of the B7-H4 antibody 20502 (afucosylated) or a humanisotype control. 24 hours post co-incubation, plates were centrifugedand supernatants were harvested and assessed for IL-2 production byAlphaLisa, according to the manufacturer's instructions.

As shown in FIG. 6B, B7-H4 demonstrated T cell checkpoint ligandactivity in this assay when endogenously expressed at physiologic levelsas IL-2 production was reduced in wells containing SK-BR-3 B7-H4+ cellsrelative to SK-BR-3 KO cells. Moreover, the B7-H4 antibody alsodemonstrated T cell checkpoint blockade activity as measured by anincrease in IL-2 production relative to isotype control treated cells(FIG. 6B). Thus, this data confirm the T cell checkpoint blockadeactivity of B7-H4 antibody 20502 (afucosylated) and show that the B7-H4antibody 20502 provides T cell checkpoint blockade activity in cellsthat endogenously express B7-H4.

Example 12 Afucosylated and Fucosylated Monoclonal Antibodies ADCCActivity

B7-H4 antibodies were assessed for ADCC activity against aB7-H4-expressing target cell line. Specifically, primary human PBMCscells were cytokine activated at 1×10⁶ cells/mL with 200 IU/mL IL-2 at37° C. The next day, cells were washed and incubated at a 40:1Effector:Target ratio with SK-BR-3 target cells that were labeled withCalcein-AM. 4 hours after incubation, target cell lysis was quantifiedusing a fluorimeter. A Triton/X treated sample served as the max lysiscontrol sample, whereas a media alone treated sample served as thebackground lysis control sample. The percent (%) specific lysis wascalculated as follows: [1-((sample− media control)/(max lysis−mediacontrol))]×100. The percent (%) specific lysis was plotted vs. antibodyconcentration and the EC50 potency was calculated using nonlinearregression curve fit (GraphPad Prism).

The B7-H4 antibodies demonstrated potent dose-dependent ADCC activityagainst the endogenous B7-H4 expressing breast cell line SK-BR-3.Moreover, the afucosylated antibodies demonstrated significantly morepotent ADCC activity in comparison to the fucosylated antibodies (FIG. 7and Table 20).

TABLE 20 ADCC activity ADCC Assay (EC50 +/− STD; nM) Antibody BINAfucosylated Fucosylated 20502 3 0.0007 +/− 1.1 × 10E−3 0.0370 +/−6.2E−2 20506 3 0.0015 +/− 2.5 × 10E−3 0.0135 +/− 2.1E−2 22213 3 0.00150.014

Example 13 Correlation of ADCC Activity with Receptor Density

B7-H4 density was quantified on the surface of SK-BR-3, HCC1569,ZR-75-1, MDA-MB-48, and HCC1964 cells by FACS according to themanufacturer's specifications. Specifically, 1×10⁵ cells were incubatedwith 15 ug/mL B7-H4 antibody on ice for 25 minutes. In parallel, onedrop of Quantum™ Simply Cellular (QSC) microspheres (pre-coated withincreasing concentrations of anti-mouse IgG capture antibody) was alsoincubated with 15 ug/mL B7-H4 antibody on ice for 25 minutes. Followingincubation, cells and QSC microspheres were pelleted and washed, andsamples were acquired on a flow cytometer. Data was analyzed using theFlowJo software. Mean fluorescence intensity (MFI) was calculated andentered into the QuickCal® spreadsheet. A regression associating eachbead's fluorescence channel value to its pre-assigned Antibody BindingCapacity (ABC) value will be calculated automatically. An ABC value wasassigned once the MFI values for the labeled cells are also added intothe template).

B7-H4 antibodies were assessed for ADCC activity against B7-H4expressing target cell lines with different levels of B7-H4 cell surfacedensity. Specifically, 1×104 SK-BR-3, HCC1569, ZR-75-1, MDA-MB-468, orHCC1964 target cells were co-incubated with dose-titrations of B7-H4antibody at 4° C. 25 minutes later, a single use vial of Jurkat-huCD16reporter cells from Promega was thawed, and 7.5×104 cells were added tothe target cell/B7-H4 antibody mixture and incubated at 37° C. 24 hourslater, the samples were brought to room temperature (RT) and incubatedwith Bio-Glo buffer. The substrate and luminescence were quantified onan EnVision multi-label reader. The data was plotted as luminescence vs.antibody concentration and the EC50 potency was calculated usingnonlinear regression curve fit (GraphPad Prism).

B7-H4 antibody ADCC activity was dependent on B7-H4 cell surfacedensity: as the numbers of cell surface molecules decreased, the amountof maximal ADCC activity also decreased. Moreover, afucosylatedantibodies demonstrated improved ADCC activity in comparison to thefucosylated antibodies, especially against target cells with lowerlevels of B7-H4 cell surface density (FIG. 8).

Example 14 In Vivo Anti-Tumor Efficacy

Seven week old female BALB/c mice were purchased from Charles RiverLaboratories (Hollister, Calif.) and were acclimated for up to threeweeks before the start of the studies. The murine colorectal carcinomacell line CT26 was engineered to express a chimeric protein consistingof the extracellular domain of murine B7-H4 with the transmembranedomain of murine B7H3. These tumor cells were implanted subcutaneouslyover the right flank of the mice at 1.0×10⁶ cells/200 μl/mouse. Prior toinoculation, the cells were cultured for no more than three passages inRPMI 1640 medium supplemented with 10% heat-inactivated Fetal BovineSerum (FBS), 2 mM L-Glutamine. Cells were grown at 37° C. in ahumidified atmosphere with 5% CO₂. Upon reaching 80-85% confluence,cells were harvested and resuspended in a 1:1 mixture of serum-free RPMI1640 and Matrigel at 5×10⁶ cells per milliliter).

Mice were monitored twice weekly following cell implantation for tumorgrowth. For tumor measurements, the length and width of each tumor wasmeasured using calipers and volume was calculated according to theformula: tumor volume (mm3)=(width (mm)×length (mm2)/2. On the day oftreatment initiation, all tumors were measured, outliers were excluded,and mice were randomly assigned to treatment groups. For anti-B7-H4treatment, antibodies used include 20502 (FIG. 9A) and 22213 (FIG. 9B).As controls, mice were administered polyclonal human IgG (Bio X Cell,BE0092) or mouse IgG2a (Bio X Cell, BE0085). The antibodies wereadministered four times via intravenous (i.v.) injection twice weeklybeginning on Day 4 or 5 after inoculation.

Tumors continued to be measured at least twice per week until tumorvolume exceeded 10% of animal weight, or approximately 2000 mm3. Thechange in tumor size is shown by graphing individual tumors relative tothe day upon which animals were inoculated with CT26 cells. P-valueswere calculated using unpaired, two-tailed t-test analyses of thecalculated tumor volumes on each day of the study. Treatment with 20502or 22213 significantly reduced tumor growth compared to human IgGcontrol (p<0.05) when administered between doses of 10-20 mg/kg (FIGS.9A and 9B).

Similar experiments were performed using mice with implanted 4T1 cells(a murine breast carcinoma cell line) or B16 (a murine melanoma cellline). These mice were treated with 20 mg/kg of a mouse surrogate of20502 called 20502-msIgG2a-F, which contains the 20502 variable regionfused to fucosylated mouse IgG2a, or with murine IgG control antibody.Treatment with 20502-msIgG2a-F significantly reduced tumor growthcompared to the murine IgG control in both the 4T1 breast carcinoma andB16 melanoma models (FIG. 10).

Additional experiments were also performed using afucosylated 20502 in aMX-1 human breast cancer xenograft model. Female NSG (NOD-scid, IL2Rgammanull) mice were purchased from the Jackson Laboratory (Bar Harbor,Me.) and were acclimated for one week before the start of the studies.The human breast cancer cell line MX-1 had previously been shown toendogenously express cell-surface B7H4 protein. These tumor cells wereimplanted subcutaneously over the right flank of the mice at 1.0×106cells/100 μl/mouse in a 1:1 mixture of serum-free RPMI 1640 and Matrigelat 5×106 cells per milliliter.

Mice were monitored twice weekly following cell implantation for tumorgrowth. For tumor measurements, the length and width of each tumor wasmeasured using calipers, and volume was calculated according to theformula: Tumor volume (mm3)=(width (mm)×length (mm)2)/2. On Day 7 afterinoculation, all tumors were measured, outliers were excluded, and micewere randomly assigned to treatment groups. All animals wereadministered a DNA construct that induced constitutive human IL-15expression in the circulation for the remainder of the study. On Day 9,half of the mice received an intravenous (i.v.) injection of 20×106human peripheral blood monocytic cells (PBMCs), acquired from StemCellTechnologies (Tukwila, Wash.). Beginning on Day 11, mice wereadministered afucosylated 20502 (20 mg/kg) or saline as a negativecontrol. Therapeutics were administered four times via intravenous(i.v.) injection twice weekly.

Tumors continued to be measured at least twice per week until tumorvolume exceeded 10% of animal weight or until the animals demonstrated a15% or greater loss of initial body weight. P-values were calculatedusing One-Way ANOVA comparing the mean tumor volumes across alltreatment groups on Day 28. As shown in FIG. 11, treatment withafucosylated 20502 significantly reduced tumor growth compared to salinecontrol (p<0.05) only when administered to mice previously injected withhuman PBMCs.

Example 15 In Vivo Anti-Tumor Efficacy in Combination with Anti-PD-1Antibody Methods

Eight week old female BALB/c mice were purchased from Charles RiverLaboratories (Hollister, Calif.) and were acclimated for up to two weeksbefore the start of the study. The murine breast carcinoma cell line 4T1was engineered to express a chimeric protein containing theextracellular domain of murine B7-H4 and the transmembrane domain ofmurine B7H3. Tumor cells were implanted orthotopically in the mammaryfat pad of the mice at 0.5×105 cells/50 μl/mouse. Prior to inoculation,the cells were cultured for no more than three passages in RPMI 1640medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS).Cells were grown at 37° C. in a humidified atmosphere with 5% CO₂. Uponreaching 80-85% confluence, cells were harvested and resuspended inserum-free RPMI 1640 on the ventral flank of each mouse into the mammaryfat pad.

Mice were monitored twice weekly following cell implantation for tumorgrowth. The length and width of each tumor was measured using calipers,and the volume was calculated according to the formula: Tumor volume(mm3)=(width (mm)×length (mm)2)/2. On the day of treatment initiation,all tumors were measured, outliers were excluded, and mice were randomlyassigned to treatment groups. For the anti-B7-H4 treatment, a mousesurrogate of 20502 called 20502-msIgG2a-F, which contains the 20502variable region fused to fucosylated mouse IgG2a, was utilized. As acontrol, mice were administered msIgG2a (anti-HEL). 20502-msIgG2a-F ormsIgG2a were administered four times via intravenous (i.v.) injectiontwice weekly beginning on Day 11 after inoculation. Anti-PD-1 (amodified version of RMP1-14 (Bio X Cell) containing a Fc silent msIgG2adomain) was administered three times via intraperitoneal (i.p.)injection twice weekly beginning on Day 11 after inoculation. Tumorscontinued to be measured at least twice per week until tumor volumeexceeded 10% of animal weight, or approximately 2000 mm³.

Results

The change in tumor size, the change in mean tumor volume, and thepercent survival are shown in FIGS. 12A, 12B and 12C, respectively.Treatment with either 20502-msIgG2a-F or anti-PD-1 significantly reducedtumor growth compared to msIgG2a control (p<0.05). The co-administrationof 20502-msIgG2a-F and anti-PD-1 significantly enhanced tumor growthinhibition compared to either monotherapy (p<0.05). Moreover,combination therapy resulted in complete tumor regression in 5 of 12mice. P-values were calculated using One-Way ANOVA analyses of thecalculated tumor volumes on each day of the study with multiplecomparisons between each group.

Example 16 Anti-B7-114 Antibody Increases NK-Cell and T-CellInfiltration and Up-Regulates PD-L1

Eight week old female BALB/c mice were purchased from Charles RiverLaboratories (Hollister, Calif.) and implanted orthotopically in themammary fat pad of the mice with 4T1+moB7-H4/H3 cells at 0.5×105cells/50 μl/mouse. On the day of treatment initiation, all tumors weremeasured, outliers were excluded, and mice were randomly assigned totreatment groups. Mice were administered huIgG1 (anti-HEL) orafucosylated 20502 at 20 mg/kg via intravenous (i.v.) injection twice(Day 11 and Day 14 post-inoculation).

At 24 hours after the second dose administration, mice were euthanizedand perfused with phosphate-buffered saline (PBS). Tumors were thenextracted, fixed in 10% formalin for five hours, rinsed in PBS, andplaced in a 30% sucrose solution for at least 24 hours. Tumors wereembedded in Tissue-Tek® O.C.T. Compound and sectioned at 20 μm in a −15°C. chamber (cryostat). Tissue-mounted slides were rinsed in 0.3% Tritonin PBS, blocked with 5% normal goat serum, and stained with primaryantibody (anti-NKp46, anti-CD3, or anti-PD-L1; 1:500) overnight. Twoserial sections from each tumor were stained.

Following overnight incubation, slides were rinsed in 0.3% Triton thenincubated with secondary antibody (1:400) for three hours in a darkhumid chamber and rinsed. The tissue was then fixed in 1%paraformaldehyde and rinsed in PBS. Coverslips were mounted usingVectashield® with DAPI, sealed with Cytoseal™, and left to dry in a darkchamber. Images were acquired manually with a fluorescence microscopeand camera. As shown in FIG. 13, treatment with afucosylated 20502resulted in increased natural killer (NK) cells (as measured by theanti-NKp46 antibody) and increased CD3+ T cells at tumor edges with amoderate increase of CD3+ T cells in the tumor core. Increased PD-L1expression was also observed.

Example 17 Anti-B7-114 Antibody Significantly Reduces Tumor Growth InVivo in 4T1- and B16-moB7-H4/H3 Models in a Dose Dependent Manner

Six- to eight-week old female BALB/c or C57B1/6 mice were purchased fromCharles River Laboratories (Hollister, Calif.) and were acclimated forup to three weeks before the start of the studies. The murine breastcarcinoma cell line 4T1 and melanoma cell line B16-F10 were engineeredto express murine a chimeric protein consisting of the extracellulardomain of murine B7-H4 with the transmembrane domain of murine B7-H3.These tumor cells were implanted orthotopically in the mammary fat padat 0.05×10⁶ cells/50 μl/mouse for 4T1+moB7-H4/H3 or subcutaneously overthe right flank of the mice at 0.5×10⁶ cells/100 μl/mouse forB16+moB7-H4/H3. Prior to inoculation, the cells were cultured for nomore than three passages in RPMI-1640 or DMEM medium supplemented with10% heat-inactivated Fetal Bovine Serum (FBS) and 2 mM L-Glutamine.Cells were grown at 37° C. in a humidified atmosphere with 5% CO₂.

Mice were monitored twice weekly following cell implantation for tumorgrowth. For tumor measurements, the length and width of each tumor wasmeasured using calipers, and volume was calculated according to theformula: Tumor volume (mm3)=(width (mm)×length (mm)2)/2. On the day oftreatment initiation, all tumors were measured, outliers were excluded,and mice were randomly assigned to treatment groups. Mice wereadministered 20502-msIgG2a-F antibody (anti-B7-H4, msIgG2a, fucosylated(also called “cmFPA150-F”) or a mouse IgG2a control antibody four timesvia intravenous (i.v.) injection twice weekly beginning on Day 11(4T1+moB7-H4/H3) or Day 6 (B16+moB7-H4/H3) after inoculation, except forthe 20 mg/kg group, which was administered continuously twice weeklyuntil the end of study.

Tumors continued to be measured at least twice per week until tumorvolume exceeded 10% of animal weight, or approximately 2000 mm3. Thechange in tumor size is shown by graphing mean tumor volume relative tothe day upon which animals were inoculated. Treatment with20502-msIgG2a-F significantly reduced tumor growth compared to mouseIgG2a control (p<0.05) when administered at doses of 1 mg/kg or greaterin the 4T1+moB7-H4/H3 model (see FIGS. 14A and 14B) and at doses of 3mg/kg or greater in the B16+moB7-H4/H3 model (FIGS. 15A and 15B).Statistical significance was calculated using OneWay ANOVA comparing alltreatment groups to msIgG2a.

FIGS. 14A and 14B show that 20502-msIgG2a-F antibody significantlyreduces growth of 4T1 expressing B7-H4/H3. BALB/c mice were inoculatedorthotopically with 4T1 breast carcinoma cells engineered to expressmurine B7-H4 ECD fused to B7-H3™. Mice were administered 20502-msIgG2a-Fantibody (anti-B7-H4, msIgG2a, fucosylated) twice weekly beginning onDay 11 after inoculation. 20502-msIgG2a-F antibody demonstrated adose-dependent reduction in tumor growth, with significant tumor growthinhibition at 30 mg/kg (p=0.0003), 20 mg/kg (p=0.0103), 10 mg/kg(p=0.0419), 3 mg/kg (p=0.0277), and 1 mg/kg (p=0.0333) as assessed byOneWay ANOVA on Day 30.

FIGS. 15A and 15B show that 20502-msIgG2a-F antibody significantlyreduces growth of B16 expressing B7-H4/H3. C57B1/6 mice were inoculatedsubcutaneously with B16-F10 melanoma cells engineered to express murineB7-H4 ECD fused to B7-H3™. Mice were administered 20502-msIgG2a-Fantibody (anti-B7-H4, msIgG2a, fucosylated) twice weekly beginning onDay 6 after inoculation. 20502-msIgG2a-F antibody demonstrated adose-dependent reduction in tumor growth (FIG. 15A), with significanttumor growth inhibition at 30 mg/kg (p=0.0085), 20 mg/kg (p=0.0041), 10mg/kg (p=0.0017), and 3 mg/kg (p=0.0420) as assessed by OneWay ANOVA onDay 23 (FIG. 15B).

Thus, the 20502-msIgG2a-F antibody significantly reduced tumor size in adose-dependent manner in two different cancer models. This data inbreast carcinoma and melanoma cell lines indicate that the20502-msIgG2a-F antibody can be used to treat patients with cancer.

Sequence Tables

GITR Antibody Sequences

SEQ ID NO Sequence 411 SGSVFSIDAM 412 LSGISSAK 413 YADVSTGWGRDAHGYW 414EVQLLESGGGEVQPGGSLRLSCAASGSVFSIDAMGWYRQAPGKQRELVAVLSGISSAKYAASAPGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYADV STGWGRDAHGYWGQGTLVTV415 EVQLLESGGGEVQPGGSLRLSCAASGSVFSIDAMGWYRQAPGKQRELVAVLSGISSAKYAASAPGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYADVSTGWGRDAHGYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGSVFSIDAMGWYRQAPGKQRELVAVLSGISSAKYAASAPGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYADVSTGWGRDAHGYWGQGTLVTVKPGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 416 EPKSSDKTHTCPPC 417 DKTHTCPPC 418ESKYGPPCPPC 419 GGSGGS 420 GGSGGSGGS 421 GGSGGSGGSGGS 422GGSGGSGGSGGSGGS 423 GGGG 424 GGGGG 425 GGGGGGCD80 Sequences

SEQ ID NO Sequence 426 VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFN WNTTKQEHFPDN 427VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 428VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNEPKSSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKCSFR1 Sequences

SEQ ID NO Sequence 429 QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQAPGQGLEWMGD INPYNGGTTF NQKFKGRVTI TADKSTSTAYMELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SS 430EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWYQQKPGQAPRL LIYAASNLES GIPARFSGSG SGTDFTLTISSLEPEDFAVY YCHLSNEDLS TFGGGTKVEI K 431 GYTFTDNYMI 432 DINPYNGGTT FNQKFKG433 ESPYFSNLYV MDY 434 KASQSVDYDG DNYMN 435 AASNLES 436 HLSNEDLST 437QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQAPGQGLEWMGD INPYNGGTTF NQKFKGRVTI TADKSTSTAYMELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTVSSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVTVSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWYVDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKEYKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEMTKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 438EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWYQQKPGQAPRL LIYAASNLES GIPARFSGSG SGTDFTLTISSLEPEDFAVY YCHLSNEDLS TFGGGTKVEI KRTVAAPSVFIFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQSGNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGECPD-1 (Nivolumab) Antibody Sequences

SEQ ID NO: Description Sequence 440 heavyQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYA chainDSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS variable region 441heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGchain LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLconstant FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVregion SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 442 lightEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR chainFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK variable region 443 lightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS chainKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC constant region 444 heavyQVQLVESGGGVVQPGRSLRLDCKASGITFS FR1 445 heavy NSGMH CDR1 446 heavyWVRQAPGKGLEWVA FR2 447 heavy VIWYDGSKRYYADSVKG CDR2 448 heavyRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT FR3 449 heavy NDDY CDR3 450 heavyWGQGTLVTVSS FR4 451 light FR1 EIVLTQSPATLSLSPGERATLSC 452 lightRASQSVSSYLA CDR1 453 light FR2 WYQQKPGQAPRLLIY 454 light DASNRAT CDR2455 light FR3 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC 456 light QQSSNWPRT CDR3457 light FR4 FGQGTKVEIK

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entirety andfor all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

Other embodiments are within the following claims.

What is claimed:
 1. An isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4, comprising heavy chainvariable region (VH) complementarity determining region (CDR) 1, VHCDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3sequences selected from the group consisting of: (a) SEQ ID NOs:458-463,respectively; (b) SEQ ID NOs:5-10, respectively; (c) SEQ ID NOs:15-20,respectively; (d) SEQ ID NOs:25-30, respectively; (e) SEQ ID NOs:35-40,respectively; and (f) SEQ ID NOs:45-50, respectively.
 2. An isolatedantibody or antigen-binding fragment thereof that specifically binds tohuman B7-H4, comprising a heavy chain variable region and a light chainvariable region comprising the amino acid sequences of: (a) SEQ IDNOs:464 and 42, respectively; (b) SEQ ID NOs:11 and 12, respectively;(c) SEQ ID NOs:21 and 22, respectively; (d) SEQ ID NOs:31 and 32,respectively; (e) SEQ ID NOs:41 and 42, respectively; or (f) SEQ IDNOs:51 and 52, respectively.
 3. The antibody or antigen-binding fragmentthereof of claim 1, wherein the antibody or antigen-binding fragmentfurther comprises a heavy chain constant region.
 4. The antibody orantigen-binding fragment thereof of claim 3, wherein the heavy chainconstant region is selected from the group consisting of humanimmunoglobulins IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chainconstant regions.
 5. The antibody or antigen-binding fragment thereof ofclaim 3, wherein the antibody or antigen-binding fragment furthercomprises a light chain constant region.
 6. The antibody orantigen-binding fragment thereof of claim 5, wherein the light chainconstant region is selected from the group consisting of humanimmunoglobulins IgGκ and IgGλ, light chain constant regions.
 7. Theantibody or antigen-binding fragment thereof of claim 1, wherein theantibody or antigen-binding fragment thereof comprises a heavy chain andlight chain comprising the amino acid sequences of: (a) SEQ ID NOs:469and 44, respectively; (b) SEQ ID NOs:13 and 14, respectively; (c) SEQ IDNOs:23 and 24, respectively; (d) SEQ ID NOs:33 and 34, respectively; (e)SEQ ID NOs:43 and 44, respectively; or (f) SEQ ID NOs:53 and 54,respectively.
 8. An isolated antibody or antigen-binding fragmentthereof that specifically binds to human B7-H4, wherein the antibody orantigen-binding fragment thereof comprises the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 of an antibody comprising a heavychain and a light chain comprising the amino acid sequence of: (a) SEQID NOs:469 and 44, respectively; (b) SEQ ID NOs:13 and 14, respectively;(c) SEQ ID NOs:23 and 24, respectively; (d) SEQ ID NOs:33 and 34,respectively; (e) SEQ ID NOs:43 and 44, respectively; or (f) SEQ IDNOs:53 and 54, respectively.
 9. The antibody or antigen-binding fragmentthereof of claim 8, wherein the CDRs are the Kabat-defined CDRs, theChothia-defined CDRs, or the AbM-defined CDRs.
 10. The antibody orantigen-binding fragment thereof of claim 1, wherein the antibody orantigen-binding fragment thereof is a human antibody, a chimericantibody, or an antigen-binding fragment thereof.
 11. The antibody orantigen-binding fragment thereof of claim 1, wherein contacting a cellexpressing B7-H4 with the antibody or antigen-binding fragment thereof:(a) induces T cell proliferation when the B7-H4 cell is in a populationof cells comprising T cells, (b) induces CD4+ T cell proliferation whenthe B7-H4 cell is in a population of cells comprising T cells, (c)induces CD8+ T cell proliferation when the B7-H4 cell is in a populationof cells comprising T cells, (d) is capable of inducing antibodydependent cell mediated cytotoxicity (ADCC) in a B7-H4-expressing cell,(e) inhibits tumor growth in a murine CT26 colorectal carcinoma model, amurine breast carcinoma 4T1 model, or a melanoma cell lineB16-moB7-H4/H3 model, and/or (f) inhibits T cell checkpoint activity ofB7-H4.
 12. The antibody of antigen-binding fragment thereof of claim 1,wherein contacting a cell expressing B7-H4 with the antibody orantigen-binding fragment: (a) increases T cell proliferation by at least5% as compared to treatment with a control antibody when the B7-H4 cellis in a population of cells comprising T cells, (b) increases CD4+ Tcell proliferation by at least 5% as compared to treatment with acontrol antibody when the B7-H4 cell is in a population of cellscomprising T cells, (c) increases CD8+ T cell proliferation by at least5% as compared to treatment with a control antibody when the B7-H4 cellis in a population of cells comprising T cells, (d) induces specificlysis in at least 20% of B7-H4 expressing cells, and/or (e) reducestumor growth by at least 25% as compared to treatment with a controlantibody.
 13. The antibody or antigen-binding fragment thereof of claim11, wherein the induction of T cell proliferation, the induction of CD4+T cell proliferation, the induction of CD8+ T cell proliferation, theADCC activity, the inhibition of tumor growth, and/or the inhibition ofT cell checkpoint activity is dose-dependent.
 14. The antibody orantigen-binding fragment thereof of claim 1, wherein the antibody orantigen-binding fragment thereof binds to cynomolgus monkey, rat, and/ormouse B7-H4.
 15. The antibody or antigen-binding fragment thereof ofclaim 1, wherein the antibody or antigen-binding fragment thereof bindsto the IgV domain of human B7-H4.
 16. The antibody or antigen-bindingfragment thereof of claim 1, wherein the antibody or antigen-bindingfragment thereof is afucosylated.
 17. The antibody or antigen-bindingfragment thereof of claim 1, further comprising a detectable label. 18.An isolated polynucleotide comprising a nucleic acid molecule encodingthe heavy chain variable region and/or the light chain variable regionof claim
 1. 19. An isolated vector comprising the polynucleotide ofclaim
 18. 20. A host cell comprising the polynucleotide of claim
 18. 21.A method of producing an antibody or antigen-binding fragment thereofthat binds to human B7-H4 comprising culturing the host cell of claim 20so that the nucleic acid molecule is expressed and the antibody orantigen-binding fragment thereof is produced.
 22. An isolated antibodyor antigen-binding fragment thereof that specifically binds to humanB7-H4 and is encoded by the polynucleotide of claim
 18. 23. Apharmaceutical composition comprising the antibody or antigen-bindingfragment thereof of claim 1 and a pharmaceutically acceptable excipient.24. A pharmaceutical composition comprising (i) antibodies orantigen-binding fragments of claim 1 and (ii) a pharmaceuticallyacceptable excipient, wherein at least 95% of the antibodies orantigen-binding fragments thereof in the composition are afucosylated.25. The pharmaceutical composition of claim 23, further comprising ananti-PD-1 antibody or an antigen-binding fragment thereof or ananti-PD-L1 antibody or an antigen-binding fragment thereof.
 26. A methodfor inducing T cell proliferation comprising contacting a cellexpressing B7-H4 with the antibody or antigen-binding fragment thereofof claim 1, wherein the B7-H4 expressing cell is in a population ofcells comprising T cells.
 27. A method for inducing CD4+ T cellproliferation comprising contacting a cell expressing B7-H4 with theantibody or antigen-binding fragment thereof of claim 1, wherein theB7-H4 expressing cell is in a population of cells comprising T cells.28. A method for inducing CD8+ T cell proliferation comprisingcontacting a cell expressing B7-H4 with the antibody or antigen-bindingfragment thereof of claim 1, wherein the B7-H4 expressing cell is in apopulation of cells comprising T cells.
 29. A method for killing a cellexpressing B7-H4 comprising contacting the cell with the antibody orantigen-binding fragment thereof of claim
 1. 30. A method for depletingB7-H4-expressing cells from a population of cells comprising contactingthe population of cells with the antibody or antigen-binding fragmentthereof of claim
 1. 31. The method of claim 26, further comprisingcontacting the cell expressing B7-H4 with an anti-PD-1 antibody or anantigen-binding fragment thereof or an anti-PD-L1 antibody or anantigen-binding fragment thereof.
 32. A method of treating a B7-H4expressing cancer in a subject, the method comprising administering tothe subject an effective amount of the antibody or antigen-bindingfragment thereof of claim
 1. 33. The method of claim 32, wherein thecancer is selected from the group consisting of head and neck cancer,small cell lung cancer, gastric cancer, melanoma, breast cancer, ductalcarcinoma, endometrial carcinoma, ovarian cancer, non-small cell lungcancer, pancreatic cancer, thyroid cancer, kidney cancer and bladdercancer.
 34. The method of claim 32 wherein the cancer is a PD-1inhibitor inadequate responder, and/or wherein the cancer is a PD-L1inhibitor inadequate responder, and/or wherein the cancer expresses alow level of PD-L1.
 35. The method of claim 32, further comprisingadministering to the subject an anti-PD-1 antibody or an antigen-bindingfragment thereof or an anti-PD-L1 antibody or an antigen-bindingfragment thereof.
 36. A method for detecting B7-H4 in a samplecomprising contacting said sample with the antibody or antigen-bindingfragment thereof of claim
 1. 37. A kit comprising the antibody orantigen-binding fragment thereof of claim 1, a detection reagent, andinstructions for use for detection of a B7-H4 antigen.
 38. An isolatedpolynucleotide comprising a nucleic acid molecule encoding the heavychain variable region or heavy chain of the antibody or antigen-bindingfragment thereof of claim 7 and/or encoding the light chain variableregion or light chain of the antibody or antigen-binding fragmentthereof of claim
 7. 39. The antibody or antigen-binding fragment thereofof claim 2, wherein the antibody or antigen-binding fragment furthercomprises a heavy chain constant region.
 40. The antibody orantigen-binding fragment thereof of claim 2, wherein the antibody orantigen-binding fragment thereof is afucosylated.
 41. The antibody orantigen-binding fragment thereof of claim 7, wherein the antibody orantigen-binding fragment thereof is afucosylated.
 42. A pharmaceuticalcomposition comprising the antibody or antigen-binding fragment thereofof claim 2 and a pharmaceutically acceptable excipient.
 43. Apharmaceutical composition comprising the antibody or antigen-bindingfragment thereof of claim 7 and a pharmaceutically acceptable excipient.44. The pharmaceutical composition of claim 24, further comprising ananti-PD-1 antibody, an anti-PD-L1 antibody, or an antigen-bindingfragment thereof.
 45. The antibody or antigen-binding fragment of claim39, wherein the antibody or antigen-binding fragment comprises a lightchain constant chain.